CN117751181A - Chimeric antigen receptor T cells - Google Patents

Abstract

The present invention relates to Chimeric Antigen Receptor (CAR) T cells and in particular, but not exclusively, to their use in immunotherapy, and for the treatment, prevention or amelioration of cancer (e.g. T cell lymphoma), various microbial infections (e.g. HIV and TB), and autoimmune diseases. The invention relates in particular to the use of CAR engineered mucosal associated constant T (MAIT) cells, and to novel methods for stimulating, isolating and expanding highly purified MAIT cells, which can then be engineered into such CAR-MAIT cells. The invention also relates to a method for in vitro expansion of MAIT cells.

Description

Chimeric antigen receptor T cells

The present invention relates to Chimeric Antigen Receptor (CAR) T cells and in particular, but not exclusively, to their use in immunotherapy, and for the treatment, prevention or amelioration of cancer (e.g. T cell lymphoma), various microbial infections (e.g. HIV and TB), and autoimmune diseases. The invention relates in particular to the use of CAR engineered mucosal associated constant T (MAIT) cells, and to novel methods for stimulating, isolating and expanding highly purified MAIT cells, which can then be engineered into such CAR-MAIT cells. The invention also relates to a method for in vitro expansion of MAIT cells. The invention extends to genetic constructs themselves, and to their use in the production of CAR-MAIT cells, as well as to transduced CAR-MAIT cells themselves. The invention also extends to various medical uses of the constructs and transduced CAR-MAIT cells, as well as pharmaceutical compositions comprising these constructs and CAR-MAIT cells.

T-cell lymphomas are a group of heterogeneous clinical invasive diseases including peripheral T-cell lymphomas (PTCL), such as adult T-cell leukemia/lymphomas (ATL) caused by human T-lymphovirus type I (HTLV-1), cutaneous T-cell lymphomas (CTCL), such as Szezali Syndrome (SS). T cell malignancies are more difficult to treat than B cell malignancies. ATL and SS represent a rare and often invasive type of T cell lymphoma, and there are currently not enough patients to participate in randomized trials to establish treatment criteria. Thus, common first line therapies are the same as therapies for the treatment of other types of T cell lymphomas. For example, currently licensed drugs for the treatment of T cell malignancies include chemotherapeutic drugs, biological response modifiers (e.g., interferons, bexarotene, and HDAC inhibitors), monoclonal antibodies (alemtuzumab, mo Jiamu rituximab, rituximab), hematopoietic Stem Cell Transplantation (HSCT), and in vitro photopheresis (ECP), among others. However, none of the treatment regimens is superior to the other treatment regimens in terms of their overall response rate or duration of response. Although allogeneic (HSCT) is the only potential therapeutic regimen, a large number of patients may not be suitable for HSCT due to age and complications and HSCT-related mortality. Thus, there is a need for more effective treatment because the median survival for current ATL treatments is only 8 months (Katsuya et al, 2015).

Recently FDA approved Chimeric Antigen Receptor (CAR) -based T cell therapies (CAR-T) are considered one of the most significant breakthroughs for the treatment of B cell malignancies, achieving nearly 100% remission by targeting CD19 antigen (Park et al, 2018). Although CAR-T is effective in treating B cell malignancies, a similar approach to T cell derived malignancies has not been established. As with most B cell malignancies containing the same immunoglobulin gene rearrangement (i.e., cloning) and expressing the pan B cell marker (e.g., CD 19), most (> 95%) ATL and CTCL are derived from dominant T cell clones expressing the defined T Cell Receptor (TCR) gene (i.e., cloning the TCR-Vbeta chain) and pan T helper marker CD 4. Thus, treatment of T lymphomas by monoclonal antibody (mAb) targeting pan-T cell markers (e.g., CD4 or TCR-Vb chain) has been tested, but only has achieved partial efficacy in small clinical trials.

Current CAR-T therapies are primarily based on traditional αβ T cells. However, antigen recognition by αβ T cells is severely limited by MHC, which makes it suitable for autologous therapy, but adoptive transfer of the variant is very difficult. In addition, due to insufficient tumor infiltration of αβ T cells, conventional CAR-T therapies show lower efficacy in solid tumors (such as CTCL), but have broad prospects in liquid tumor treatment. However, conventional CAR-T cell therapies have some major drawbacks that limit their further application. For example, current CAR-T therapies: (i) Limited by autotransfusion due to Graft Versus Host Disease (GVHD), (ii) limited by targeting/oncolytic toxicity of the cytokine release syndrome; (iii) Drawbacks of autologous CAR-T, such as variability of patient T cell function, product standardization and cost.

Thus, there is a need to provide improved immunotherapy for T cell malignancies (e.g., T cell lymphomas, including PTCL and CTCL) and for the treatment of microbial infections (e.g., HIV and TB).

To treat T cell malignancies, the inventors focused attention on mucosal-related constant T cells (MAIT cells), which are a subset of T cells in the immune system that exhibit innate effector-like properties. MAIT cells are defined by constant use of the T cell receptor chain V.alpha.7.2, restricted by the Major Histocompatibility Complex (MHC) -Ib associated protein MR1, and exhibit high expression of the C-type lectin CD161 and IL18 receptors. In humans, MAIT cells are present in the blood, liver, lung and mucous membranes, and can protect against microbial activity and infection. MHC class I protein MR1 is responsible for the presentation of bacterial produced vitamin B metabolites (e.g.5-OP-RU) to MAIT cells. After MR1 presents the foreign antigen, MAIT cells secrete pro-inflammatory cytokines and are able to lyse bacterially infected cells.

Current methods of expansion of MAIT cells require the use of human allogeneic feeder cells to support the growth of MAIT cells in vitro culture, which is difficult for mass production and quality control of human adaptive immunotherapy. In addition, the MAIT cells produced using these known methods contain a proportion of other cell subsets, such as conventional CD4+ T cells and CD8+ T cells, which therefore makes the resulting MAIT isolate entirely unsuitable for allogeneic adoptive transfer, as these cell subsets can lead to graft versus host disease (GvHD).

Thus, there is also a need for an improved method to stimulate and isolate pure MAIT cell cultures without the need for allogeneic feeder cells to expand the production of MAIT cells for allogeneic adoptive transfer.

As described in the examples, the inventors developed a new method for stimulating and isolating high purity MAIT cell cultures from human PBMCs. The inventors have also developed novel genetic constructs and vectors (referred to herein as "CART4" and "cartvb7.1") that encode Chimeric Antigen Receptors (CARs) and then transduce into pure MAIT cells, resulting in CAR-MAIT cells that specifically target CD4 molecules on T cells (using "CART 4") or the TCR-Vbeta 7.1 chain (using "cartvb7.1"). This is achieved by creating a novel genetic construct comprising an scFv of either: (i) An anti-CD 4 mAb (e.g., hu5A 8) or (ii) an anti-TCR-Vb 7.1mAb (e.g., 3G 5), together with a CD28/4-1BB/CD3zeta chain signaling moiety, forms a third generation CAR. These CARs were then transduced into MAIT cells purified from Peripheral Blood Mononuclear Cells (PBMCs) to produce final CAR-MAIT cells targeting CD4 on T cells or TCR-Vbeta 7.1 chains on T cells. The inventors have demonstrated that these CAR-MAIT cells surprisingly exhibit comparable or even superior anti-T lymphoma activity as conventional CAR-T cells.

Thus, in a first aspect of the invention, provided herein are mucosal associated constant T (MAIT) cells expressing a Chimeric Antigen Receptor (CAR).

As described herein, MAIT cells are non-conventional and congenital T cells expressing constant T Cell Receptors (TCRs), are highly conserved during mammalian evolution, recognize microbial antigens presented by MR1 proteins, and exist in human blood and maintain tissue homeostasis to obtain a broad range of antimicrobial responsiveness. Furthermore, it is advantageous that the antigen recognition mechanism of the MAIT cells is independent of MHC, which makes the MAIT cells a powerful candidate for allogeneic T cell killing therapy, so that it is not limited to autologous therapies, such as current MHC-dependent T cell therapies. In other words, the MAIT cells have low alloreactivity and are less prone to induce human Graft Versus Host Disease (GVHD), thus representing an ideal T cell subset for allogeneic CAR-T therapy. In contrast, CART-MAIT cells of the present invention and cell therapies resulting therefrom can readily undergo allogeneic metastasis, and the endogenous nature of the MAIT cells makes them promising candidates for infiltration into peripheral tissues for solid tumor treatment. Thus, CAR-MAIT cells can effectively penetrate into solid tumors, which makes MAIT cell-based cell therapies a promising therapy for solid and liquid tumors.

Advantageously, the CAR-MAIT cells of the present invention may be used to treat T cell malignancies, such as adult T cell leukemia/lymphoma (ATL) caused by human T lymphocyte virus type I (HTLV-1), cutaneous T Cell Lymphoma (CTCL), such as Sezary Syndrome (SS). It is understood that T cell malignancies are a group of heterogeneous clinical invasive diseases and are more refractory to treatment than B cell malignancies. Advantageously, CAR-MAIT therapy is expected to exhibit enhanced efficacy against solid tumors and is allogeneic, so that autograft is not required, thus positioning it as a ready-to-use therapy.

Preferably, in one embodiment, the CAR-MAIT cells express a CAR that targets a CD4 antigen on T cells. Thus, preferably, the CAR is specific for CD4 antigen on T cells. It will be appreciated that the CD4 antigen is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages and dendritic cells (T cell surface glycoprotein CD4[ Chiren ] UniProt No. P01730.1; NCBI reference sequence NP-000607.1). One embodiment of the polypeptide sequence of the CD4 antigen is represented herein as SEQ ID No:1, as follows:

thus, preferably, the CAR pair comprises a sequence substantially as set forth in SEQ ID No:1 or a variant or fragment thereof.

Preferably, in another embodiment, the CAR-MAIT cells express a CAR that targets the T Cell Receptor (TCR) β chain variable region (Vbeta) on T cells. It is understood that T Cell Receptors (TCRs) are protein complexes found on the surface of T cells or T lymphocytes, which are responsible for recognizing antigen fragments as peptides bound to Major Histocompatibility Complex (MHC) molecules. TCRs consist of two distinct protein chains. In humans, TCR in 95% of T cells consists of TRA-encoded alpha (α) chains and TRB-encoded beta (β) chains.

Table 1 below lists the Vbreta region on T cells and the associated coding genes, and any one or more of them can be targeted by CARs on CAR-MAIT cells of the present invention.

TABLE 1 beta chain variable region (Vbeta) on T cells

The CAR-MAIT cells may express CARs that target multiple T Cell Receptor (TCR) β chain variable regions (Vbeta) on the T cells. Preferably, the plurality of Vbeta regions may be selected from a set of Vbeta regions shown in table 1. For example, the CAR-MAIT cells may express CARs targeting at least two, at least three, or at least four T Cell Receptor (TCR) β chain variable regions (Vbeta) on T cells, preferably as shown in table 1. The CAR-MAIT cells may express CARs targeting at least five, at least six, or at least seven T Cell Receptor (TCR) β chain variable regions (Vbeta) on T cells, preferably as shown in table 1. The multiple TCR Vbeta regions may be the same or different Vbeta regions.

The following Vb family is believed to be frequently associated with T cell lymphomas, namely, vb 1, vb 2, vb3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20. Thus, in a preferred embodiment, the CAR-MAIT cell expresses a CAR targeting one or more TCR Vbeta regions on the T cell, selected from the group consisting of: vb 1, vb 2, vb3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20. Preferably, the CAR-MAIT cells express CARs targeting at least two or three TCR Vbeta regions on T cells selected from the group consisting of: vb 1, vb 2, vb3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20.

Thus, preferably, the CAR has specificity for at least one or more TCR Vbeta regions on the T cell, most preferably the TCR-Vbeta 7.1 chain. One embodiment of the polypeptide sequence of the TCR Vbase 7.1 region (Chiren rearranged TCR Vbase 7.1UniProtKB/Swiss-Prot: A0A577.1) is represented herein as SEQ ID No:2, as follows:

MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSP

ECPNSSLLNLHLHALQPEDSALYLCASSQ

[SEQ ID No:2]

thus, preferably, the CAR pair comprises a sequence substantially as set forth in SEQ ID No:2 or a variant or fragment thereof, or at least one or more TCR Vbeta regions (more preferably TCR-Vbeta 7.1 chains) thereof.

Preferably, the CAR-MAIT cells are configured to directly kill the targeted T cells by inducing apoptosis.

Preferably, the CAR-MAIT cells comprise one or more coding sequences that allow for the controlled or induced elimination of the CAR-MAIT cells, e.g. in case of adverse effects in the patient. The skilled person may refer to the one or more coding sequences as so-called "suicide genes".

Thus, in one embodiment, one or more coding sequences may encode an Epidermal Growth Factor Receptor (EGFR) or a truncated epidermal growth factor receptor (tEGFR) (reference) (UniProt No. P00533; NCBI reference sequence NP-001333826.1). Expression of tgfr can be controlled by anti-EGFR mAb (cetuximab) to monitor or eliminate CAR-T cells in patients. It is understood that EGFR is known as HER1 in humans and is a transmembrane protein, which is the receptor for the Epidermal Growth Factor (EGF) member of the extracellular protein ligand (UniProt No. P01133; NCBI reference sequence NP-001171601.1).

In another embodiment, one or more of the coding sequences may encode an inducible caspase 9 (iC 9) (mol. Therapy, diacon et al, 580,25,3,March 2017). iC9 is a modified human caspase 9 (UniProt No. p55211; NCBI reference sequence np_ 001220.2), fused to human FK506 binding protein (UniProt No. p62942; NCBI reference sequence np_ 000792.1), allowing for conditional dimerization using chemical inducers (caspase-induced drug (CID), rimiducid), triggering CAR-T cell apoptosis expressing the fusion protein.

Preferably, the CAR-MAIT cells comprise a coding sequence encoding a truncated epidermal growth factor receptor (tgfr) and/or an inducible caspase 9 (iC 9). More preferably, the CAR-MAIT cells comprise coding sequences encoding a truncated epidermal growth factor receptor (tgfr) and an inducible caspase 9 (iC 9).

It is understood that CAR-MAIT cells are produced by transducing MAIT cells with a nucleic acid or genetic construct encoding the CAR. It is important to use a culture of highly purified MAIT cells for the CAR transduction step to produce T cell specific and active CAR-MAIT cells. Accordingly, the present inventors developed an efficient method for isolating purified MAIT cells from human Peripheral Blood Mononuclear Cells (PBMC) by combining Magnetically Activated Cell Sorting (MACS) with Fluorescence Activated Cell Sorting (FACS) such that the resulting method yields a large number of MAIT cells with high expansion.

Thus, preferably, the MAIT cells are isolated from human Peripheral Blood Mononuclear Cells (PBMC). Preferably, the MAIT cells are isolated from PBMC by Magnetic Activated Cell Sorting (MACS) and/or Fluorescent Activated Cell Sorting (FACS), more preferably both MACS and FACS. The inventors believe that their MAIT separation method is novel in itself.

Thus, in a second aspect, provided herein is a method of isolating a MAIT cell, the method comprising:

(i) Providing Peripheral Blood Mononuclear Cells (PBMCs); and

(ii) The PBMCs are subjected to Magnetic Activated Cell Sorting (MACS) and/or Fluorescent Activated Cell Sorting (FACS) to separate the MAIT cells therefrom.

Preferably, the method of the invention results in the isolation of pure isolated MAIT cells. In one embodiment, the method comprises MACS or FACS of PBMCs to isolate the MAIT cells therefrom. Preferably, however, the method comprises subjecting the PBMCs to both MACS and FACS to isolate the MAIT cells therefrom. Preferably, PBMC are first MACS followed by FACS. Preferably, the method comprises isolating TCR V.alpha.7.2+ cells from PBMC by MACS, and then FACS using MR1-5-OP-RU tetramer staining to isolate MAIT cells.

MACS procedures (in step (ii)) may involve collection of PBMCs followed by washing of the cells with binding buffer. The supernatant may be discarded and the resulting cell pellet resuspended in MACS buffer (e.g., at a concentration of 1X 10 ⁷ 100 μl). The solution may be contacted (e.g., at a ratio of 1:100) with an Phycoerythrin (PE) anti-human TCR vα7.2 antibody. The solution may be mixed and then may be incubated (e.g., on ice for 30 minutes). Cells can be washed with MACS buffer (e.g., centrifuged at 300xg for 5 minutes). Cells can be resuspended in MACS buffer (e.g., at a concentration of 10 ⁷ 80 μl). The suspension may be contacted with the anti-PE microbeads, which may then be incubated (e.g., on ice for 20 minutes). Cells can be washed (e.g., 10 volumes of MACS buffer). The solution may be centrifuged (e.g., at 300x g for 5 minutes). Cells can be resuspended (e.g., in 1ml MACS buffer). The MS column can be buffered with MACSThe liquid is pre-washed and assembled on the magnet. Cells can be applied to the column and MACS performed. The column may then be washed one or more times (e.g., with MACS buffer each time). The column may be removed from the magnet and bound cells may be eluted from the column (e.g., in MACS buffer).

The FACS procedure (in step (ii)) may include collecting magnetically separated cells, followed by centrifugation (e.g. at 300x g for 5 minutes). The cells can be resuspended (e.g., with FACS buffer at 10 ⁷ Concentration re-suspension of 100 μl). The solution may be contacted with BV421 labeled human 5-OP-RU MR1 tetramer (e.g., in a ratio of 1:500) and APC-H7-conjugated anti-human CD3 (e.g., in a ratio of 1:200). The solution may then be incubated (e.g., on ice for 20 minutes). Cells can be washed (e.g., with 10 volumes of FACS buffer). The solution may be centrifuged (e.g., at 300x g for 5 minutes). Cells may be resuspended (e.g., in 2ml FACS buffer). The cell samples can then be loaded into a FACS sorter (e.g., BD Prodigy sorter) and FACS performed.

The MAIT cell is activated in a subsequent step after step (ii) prior to transduction of the isolated MAIT cell with a nucleic acid encoding a CAR, preferably in the method of the second aspect. Thus, the method further comprises activating the isolated MAIT cells with an anti-CD 3 antibody, preferably in vitro. The method comprises activating isolated MAIT cells with an anti-CD 28 antibody, preferably in vitro. Preferably, the isolated MAIT cells are activated substantially simultaneously or sequentially with anti-CD 3 and anti-CD 28 antibodies. Sequential activation may involve contacting the MAIT cell first with anti-CD 3 and then with an anti-CD 28 antibody, or first with an anti-CD 28 antibody and then with an anti-CD 3 antibody. The contacting with the antibody may last for at least one, two or three days.

The MAIT cell activation procedure may include collecting sorted MAIT cells by centrifugation (e.g., at 300x g for 5 minutes). The supernatant may be discarded and the pellet may be resuspended (e.g., to 10 in R10 medium ⁶ Individual cells/ml). The solution may be contacted with Dynabeads human T activator CD3/CD28 (e.g., by vortexing for 30 seconds). The desired volume of Dynabeads can be transferred into a test tube. An equal volume of buffer may be added to the testIn the tube and may be mixed (e.g., by vortexing for 5 seconds). The tube may be placed on a magnet (e.g., 1 minute) and the supernatant may be discarded. The tube may be removed from the magnet and the washed Dynabeads may be resuspended (e.g., in R10 medium). The desired volume of Dynabeads can be contacted with the cell suspension (e.g., 100IU/ml IL-2 in a 24 well plate in an incubator at 37 ℃ C. To obtain a microbead to cell ratio of about 1:1).

The method may then comprise transducing the isolated and now activated MAIT cell with a nucleic acid encoding a CAR.

MAIT cells are a subset of congenital T cells, defined as CD3 ⁺ TCRVa7.2 ⁺ CD161 ⁺ Cells, recognizing the MHC class I molecule MR1. Previous studies have shown that MAIT cells can be expanded in vitro, but require the presence of allogeneic feeder cells. However, one problem with this approach is that it is difficult to mass produce and quality control. As described in example 10 and shown in fig. 15, the inventors have now developed a surprisingly effective method for in vitro expansion of MAIT cells by first stimulating PBMCs with antigen (5-OP-RU) -loaded MR1 tetrameric beads or 5-OP-RU (alone), in vitro culture in the presence of various cytokine combinations for up to 6 days. MAIT cells are then isolated by MACS or FACS sorting, followed by further expansion by anti-CD 3/CD28 beads for CAR-based therapy, as described above.

Thus, in preferred embodiments, the method may comprise stimulating the PBMCs prior to subjecting the PBMCs to MACS and/or FACS steps (i.e., step ii). Preferably, the initial stimulation step comprises contacting PBMCs with: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and/or (b) a cytokine. Preferably, the stimulating step comprises contacting PBMCs: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and (b) a cytokine. The step of stimulating may comprise contacting the PBMCs with the antigen and/or cytokine for at least 1 day, 2 days, or 3 days. The stimulating step may last for at least 4 days, 5 days or 6 days. Preferably, the step of stimulating comprises contacting the PBMCs with an antigen and/or cytokine in an in vitro culture.

WO 2015/149130 describes MR1/5-OP-RU and 5-OP-RU, the entire contents of which are incorporated herein by reference. Thus, preferably, the antigen comprises MR1/5-OP-RU or 5-OP-RU, as described in WO 2015/1491130 (PCT/AU 2015/050148).

The cytokine may be any interleukin. Preferably, however, the cytokine may be one or more interleukins selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-23 or any combination thereof. The concentration of the interleukin may be at least 5ng/ml, 10ng/ml or 20ng/ml, preferably at least 30ng/ml, 40ng/ml or 50ng/ml.

For example, the one or more interleukins may comprise: (i) IL-2 alone (condition 1 in fig. 15); (ii) IL-12 and IL-18 (condition 11 in FIG. 15); (iii) IL-2, IL-12 and IL-18 (condition 3 in FIG. 15); (iv) IL-12, IL-18 and IL-23 (condition 12 in FIG. 15); (v) IL-2, IL-12, IL-18 and IL-23 (condition 13 in FIG. 15), or (vi) IL-7, IL-15, IL-12 and IL-18 (condition 8 in FIG. 15).

Most preferably, the one or more interleukins may comprise a combination of IL-12, IL-18 and IL-23. Thus, preferably, the stimulating step comprises contacting the PBMCs with: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and (b) IL-12, IL-18 and IL-23 combination.

The inventors believe that they have devised a new method of stimulating MAIT cells in PBMC cultures.

Thus, in another aspect, provided herein is a method of stimulating a MAIT cell in a PBMC culture, the method comprising contacting a PMBC culture with: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and/or (b) one or more interleukins selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-23, or any combination thereof.

Preferably, the one or more interleukins is IL-12, IL-18 and/or IL-23. More preferably, the one or more interleukins are IL-12, IL-18 and IL-23.

The inventors believe that their method of producing CAR-MAIT cells is novel in itself.

Accordingly, in a third aspect, provided herein is a method of producing a CAR-MAIT cell, the method comprising:

(i) Providing Peripheral Blood Mononuclear Cells (PBMCs);

(ii) MACS and/or FACS are performed on PBMCs to isolate MAIT cells therefrom;

(iii) Activating the isolated MAIT cells, optionally by contacting them with an anti-CD 3 and/or anti-CD 28 antibody; and

(iv) Transduction of activated MAIT cells with nucleic acid encoding a CAR, thereby producing CAR-MAIT cells.

Steps (i), (ii) and/or (iii) of the method of the third aspect may be the same as those described herein with respect to the method of the second aspect, and so these method steps are interchangeable.

Further, preferably, the method comprises stimulating the PBMCs prior to subjecting the PBMCs to MACS and/or FACS steps (i.e. step ii). Preferably, the initial stimulation step comprises contacting PBMCs with: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and/or (b) a cytokine. Preferably, the stimulating step comprises contacting PBMCs: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and (b) a cytokine. The cytokine may be an interleukin as described in relation to the second aspect, preferably one or more interleukins selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-23 or any combination thereof, as described above.

Most preferably, the one or more interleukins may comprise a combination of IL-12, IL-18 and IL-23. Thus, preferably, the stimulating step comprises contacting the PBMCs with: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and (b) IL-12, IL-18 and IL-23 combination.

Preferably, the MAIT cell is activated in step (iii) prior to transduction of the isolated MAIT cell with a nucleic acid encoding a CAR in step (iv). Isolated MAIT cells may be activated with an anti-CD 3 antibody, preferably in vitro. Isolated MAIT cells may be activated with an anti-CD 28 antibody, preferably in vitro. Preferably, the isolated MAIT cells are activated substantially simultaneously or sequentially with anti-CD 3 and anti-CD 28 antibodies as described in relation to the method of the second aspect.

The MAIT cell transduction sequence (i.e., step (iv)) may comprise transducing the MAIT cell with a nucleic acid encoding a CAR. The transduction step may comprise viral transduction. Preferably, the MAIT cell transduction sequence comprises transduction of the MAIT cell with a nucleic acid encoding a CAR by a retrovirus. The MAIT cells may be transduced with any nucleic acid encoding a CAR as described herein, e.g., a vector according to the fifth aspect or according to the sixth aspect. The nucleic acid can encode a CAR targeting CD4 antigen or at least one or more of the Vbeta regions of the TCR on T cells. Preferably, the nucleic acid may encode a CAR that targets one or more of the TCR Vbeta regions (and more preferably the TCR-Vbeta 7.1 chain) shown in table 1 above the T cell. The nucleic acid may encode a CAR targeting one or more TCR Vbeta regions on a T cell, the TCR Vbeta regions selected from the group consisting of: vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20.

Preferably, transduction is performed at least 34 hours, 36 hours, or 48 hours after activation of the MAIT cell. Prior to transduction (e.g., about 1 day ago), retroNectin coated plates may be prepared. RetroNectin (e.g., about 15 μg) may be contacted with PBS (e.g., about 1 ml) to form a solution. The method may include introducing the solution into one well of a 24-well plate that has not been treated with tissue culture. The plates may be wrapped (e.g., with a preservative film) and stored at about 4℃ (e.g., overnight in a refrigerator). On the day of gene transfer, unbound retroNectin was removed from the wells. Wells may be washed (e.g., one or two washes with 2ml PBS). Preferably, the wells are not allowed to dry. Retrovirus supernatant can be thawed (e.g., in a 37 ℃ water bath). Preferably, the viral supernatant (e.g., about 1 ml) is transferred to each well of a RetroNectin coated plate. The plate may be wrapped with a preservative film. Plates may be centrifuged (e.g., at 1000x g for 2 hours at 32 ℃). Upon centrifugation, activated MAIT cells may be collected. The harvested cells can be resuspended (e.g.in fresh R10 medium containing 100IU/ml IL-2 to a concentration of 1X10 ⁶ /ml). After centrifugation is complete, the supernatant can be discarded from the plate. A cell suspension (e.g. 1 ml) may be added to each well. The plate may be centrifuged (e.g., at 500x g for 10 minutes). The plates may then be incubated (e.g., in an incubator at 37 ℃). The transduction step may be repeated if desired to obtain higher transduction efficiencies. Transduction efficiencies can be measured by flow cytometry about 48 hours after transduction.

In the method of the third aspect, the method of the invention preferably comprises the steps ofAmplifying the CAR-MAIT cells in a subsequent step after step (iv). The CAR-MAIT cell expansion step may comprise harvesting the transduced CAR-MAIT cells one or two days after transduction, preferably using a retrovirus or virus. The harvested cells may be counted (e.g., by a cytometer). The harvested cells can then be transferred into wells of a plate (e.g., 1x 10 ⁷ Individual cells were transferred to wells of Grex6M well plates). The harvested cells may then be contacted with an interleukin. For example, the interleukin may be IL-2 (e.g., about 100 IU/ml) contained in a suitable medium, such as R10 medium (e.g., 130 ml). The plate may be returned to the incubator. IL-2 may then be updated (e.g., to a final concentration of 100IU/ml every three days). After 8-12 days of culture, the CAR-MAIT cells can be harvested. The amplified CAR-MAIT cells may be used for phenotypic testing, functional analysis, and/or freezing for later use (e.g., in liquid nitrogen).

Advantageously, as described in the examples, 11 days of culture produced 100-fold expansion levels of CAR-MAIT cells with transduction efficiencies greater than 50%. The inventors have observed that CAR-MAIT cells surprisingly exhibit a cytotoxic efficacy at least comparable to cd8+ T cells expressing conventional CARs.

In a fourth aspect, provided herein is a CAR-MAIT cell obtained or obtainable by the method of the third aspect.

As described herein, the isolated MAIT cells obtained using the methods of the second or third aspects may be activated and ultimately transduced with a nucleic acid construct encoding a CAR to produce the CAR-MAIT cells of the first or fourth aspects. As described herein, the inventors developed novel genetic constructs and recombinant vectors encoding CARs that specifically target the CD4 molecule (in which case the constructs and vectors are referred to herein as "CART 4") or one or more TCR-Vbeta regions, e.g., the TCR-Vbeta 7.1 chain on T cells (in which case the constructs and vectors are referred to herein as "cartvb7.1"). Any of these constructs and vectors may be used to transduce the MAIT cells in the methods of the third or fourth aspects.

The genetic construct comprises an scFv of any one of: (i) An anti-CD 4 mAb (e.g., hu5A 8) or (ii) an anti-TCR-Vb mAb, e.g., an anti-TCR-Vb 7.1mAb (e.g., 3G 5), forms a third generation CAR with a CD28/4-1BB/CD3zeta chain signaling moiety. The CAR-encoding construct further comprises at least one safety switch encoded by a so-called suicide gene, such as truncated epidermal growth factor receptor (tgfr) and/or inducible caspase 9 (iC 9), and which is capable of clearing the resulting CAR-T cells, thus providing a sophisticated monitoring system or safety mechanism when CAR-T cells are used in therapy. Expression of tgfr can be recognized by anti-EGFR mabs (e.g., cetuximab) for monitoring or eliminating CAR-T cells, whereas iC9 is a modified human caspase 9 fused to a human FK506 binding protein, and can be conditionally dimerized using a dimerization chemistry inducer (e.g., rimiducid) to trigger apoptosis of CAR-T cells expressing the fusion protein. The inventors believe that the CAR-encoding nucleic acid constructs they develop are novel in themselves. Referring to fig. 1A (1 and 2), this schematic diagram illustrates the functional elements comprised in both embodiments of the CAR coding construct according to the invention, i.e. fig. 1A (1) shows "CART4" and fig. 1A (2) shows "cartvb7.1". However, it should be appreciated that "cartvb7.1" having an anti-TCR-Vb 7.1 targeting the Vbeta 7.1 family is purely illustrative, any Vbeta region may be targeted by the CAR, such as any Vbeta shown in table 1, in particular any of Vb 1, vb 2, vb 3, vb 5.1, vb7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20.

Thus, in a fifth aspect, provided herein is a kit comprising a promoter operably linked to a first coding sequence encoding an anti-CD 4 Chimeric Antigen Receptor (CAR) or an anti-T Cell Receptor (TCR) Vbeta CAR.

The promoter may be any suitable promoter, including constitutive, active, inducible or tissue-specific. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the Cytomegalovirus (CMV) promoter, human elongation factor-1 alpha (hEGF), ubiquitin C promoter (Ubic), phosphoglycerate kinase Promoter (PGK), simian virus 40 early promoter (SV 40), and chicken beta-actin promoter coupled to the CMV early enhancer (CAGG). Inducible promoters belong to the category of regulatory promoters. The inducible promoter may be induced by one or more conditions, such as physical conditions, the microenvironment of the engineered immune effector cell or the physiological state of the engineered immune effector cell, an inducer (i.e., an inducer), or a combination thereof. In some embodiments, the induction conditions do not induce expression of the endogenous gene in the engineered mammalian cells and/or in the subject receiving the pharmaceutical composition. In some embodiments, the induction conditions are selected from the following: inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox status, tumor environment, and activation status of the engineered mammalian cells.

In one embodiment, the promoter may be a PGK promoter (EMBL NO: A19297.1). In one embodiment, the PGK promoter is referred to herein as SEQ ID No:3, as follows:

GGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCT

CTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCT

CCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCG

TGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTG

GGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGA

GGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATTCTCCGGGCCTTTCG

[SEQ ID No:3]

thus, preferably, the promoter may comprise a nucleotide sequence substantially as set forth in SEQ ID No:3 or a fragment or variant thereof.

The nucleic acid construct may comprise a nucleotide sequence encoding a signal peptide. Advantageously, the signal peptide is configured to direct the CAR (i.e., it is a fusion protein) to the T cell outer membrane. Preferably, the sequence encoding the signal peptide is located 3' of the promoter. Preferably, the signal peptide is an igκ signal peptide. In one embodiment, the signal peptide may have a sequence referred to herein as SEQ ID No:4, as follows:

METDTLLLWVLLLWVPGSTGD

[SEQ ID No:4]

thus, preferably, the construct comprises a coding sequence having a sequence substantially as set forth in SEQ ID No:4 or a fragment or variant thereof.

In one embodiment, the nucleotide sequence encoding the signal peptide is referred to herein as SEQ ID No:5, as follows:

ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGAC

[SEQ ID No:5]

thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:5 or a fragment or variant thereof.

Preferably, the first coding sequence is located 3' to the sequence encoding the signal peptide. In a first embodiment of the nucleic acid construct, the first coding sequence encodes an anti-CD 4 Chimeric Antigen Receptor (CAR). Preferably, the CAR pair comprises a sequence substantially as set forth in SEQ ID No:1 or a variant or fragment thereof.

As shown in fig. 1A (1) ("CART 4"), the first coding sequence may encode an scFv region, which may comprise a VL (variable light chain) sequence and a VH (variable heavy chain) sequence. Preferably, the VL sequence is located upstream (i.e. 5') of the VH sequence. However, in some embodiments, the VH sequence may be located upstream of the VL sequence.

In one embodiment, the VL and VH sequences may be Hu5A8 (i.e., hybridoma clone name of anti-CD 4 monoclonal antibody) light chain variable region and heavy chain variable region for binding to CD4 antigen on T cells.

Thus, in one embodiment, the first coding sequence (which may encode a VL sequence for binding CD 4) encodes a sequence referred to herein as SEQ ID No:6, as follows:

DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQ

AEDVAVYYCQQYYSYRTFGGGTKLEIK

[SEQ ID No:6]

thus, preferably, the first coding sequence comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:6 or a fragment or variant thereof.

In one embodiment, the first coding sequence (which may encode a VL sequence for binding CD 4) comprises a sequence referred to herein as SEQ ID No:7, as follows:

GACATTGTGATGACTCAGAGCCCCGACAGCCTGGCCGTCTCACTGGGCGAAAGGGTGACCATGAATTGTAAATCTTCTCAGAGCC

TGCTGTACAGTACAAACCAGAAAAATTACCTGGCCTGGTATCAGCAGAAACCCGGCCAGAGCCCTAAGCTGCTGATCTATTGGGC

AAGTACCCGAGAGTCAGGAGTGCCAGACAGATTCTCCGGGTCTGGAAGTGGCACAGACTTCACCCTGACAATTAGCTCCGTGCAG

GCCGAGGACGTGGCTGTCTACTATTGCCAGCAGTACTATAGCTACCGAACTTTCGGCGGGGGAACCAAACTGGAAATCAAG

[SEQ ID No:7]

thus, preferably, the first coding sequence comprises a sequence substantially as set forth in SEQ ID No:7 or a fragment or variant thereof.

In one embodiment, the first coding sequence (which may encode a VH sequence for binding CD 4) encodes a sequence referred to herein as SEQ ID No:8, as follows:

QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSS

LRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS

[SEQ ID No:8]

Thus, preferably, the first coding sequence comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:8 or a fragment or variant thereof.

In one embodiment, the first coding sequence (which may encode a VH sequence for binding CD 4) comprises a sequence referred to herein as SEQ ID No:9, as follows:

CAGGTGCAGCTGCAGCAGTCCGGACCAGAGGTGGTCAAACCCGGCGCTAGCGTCAAAATGTCCTGTAAGGCATCTGGCTACACTT

TCACCTCTTATGTGATTCACTGGGTCAGACAGAAGCCTGGGCAGGGACTGGACTGGATCGGGTACATTAACCCATATAATGATGG

AACTGACTACGATGAAAAGTTTAAAGGCAAGGCCACACTGACTTCCGACACCTCAACAAGCACTGCTTATATGGAGCTGTCTAGT

CTGAGGTCTGAAGACACAGCAGTGTACTATTGCGCCCGCGAGAAGGATAACTACGCCACTGGCGCTTGGTTTGCATATTGGGGCC

AGGGGACCCTGGTGACAGTCTCATCC

[SEQ ID No:9]

thus, preferably, the first coding sequence comprises a sequence substantially as set forth in SEQ ID No:9 or a fragment or variant thereof.

Preferably, the VH (e.g., SEQ ID No: 9) and VL (e.g., SEQ ID No: 7) sequences, when in either orientation, are separated by a linker sequence. In one embodiment, the linker sequence may be a G4S linker sequence, which is referred to herein as SEQ ID No:10, as follows:

GGGGSGGGGSGGGGS

[SEQ ID No:10]

thus, preferably, the first coding sequence comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:10 or a fragment or variant thereof.

In one embodiment, the linker sequence may consist of the sequence referred to herein as SEQ ID No:11, as follows:

GAGGAGGAGGCAGTGGCGGAGGAGGGTCAGGAGGAGGAGGAAGC

[SEQ ID No:11]

thus, preferably, the linker sequence comprises a sequence substantially as set forth in SEQ ID No:11 or a fragment or variant thereof.

However, in a second embodiment of the nucleic acid construct ("cartvb7.1"), the first coding sequence encodes an anti-T Cell Receptor (TCR) Vbeta region CAR. It should be appreciated that any Vbeta region can be targeted by the CAR. For example, any Vbeta region listed in table 1 can be targeted by a CAR encoded by the first coding sequence.

The first coding sequence may encode a plurality of T Cell Receptor (TCR) β chain variable region (Vbeta) CARs. Preferably, the plurality of Vbeta regions may be selected from a set of Vbeta regions shown in table 1. Two or more Vbeta region-targeting CARs may also be combined on the same construct. For example, the construct may comprise a coding sequence encoding at least two, at least three, or at least four T Cell Receptor (TCR) β chain variable regions (Vbeta) targeting CARs, preferably as shown in table 1. The construct may comprise a coding sequence encoding at least five, at least six, or at least seven T Cell Receptor (TCR) β chain variable regions (Vbeta) targeting CARs, preferably as listed in table 1. The multiple TCR Vbeta regions may be the same or different Vbeta regions.

The following Vb family is believed to be frequently associated with T cell lymphomas, namely, vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20. Thus, in a preferred embodiment, the construct comprises a coding sequence encoding at least one CAR that targets one or more TCR Vbeta regions on a T cell selected from the following Vbeta regions: vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20. Preferably, the construct comprises a coding sequence encoding at least one CAR that targets at least two or three TCR Vbeta regions on a T cell selected from the following Vbeta regions: vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20.

Thus, preferably, provided herein is a nucleic acid construct comprising a promoter operably linked to a first coding sequence, wherein the first coding sequence encodes a plurality of anti-T Cell Receptor (TCR) Vbeta CARs, wherein different Vbeta region cells on the T Cell Receptor (TCR) Vbeta CARs are targeted.

Preferably, the CAR has specificity for the TCR Vbeta region (preferably the TCR-Vbeta 7.1 chain) comprising a sequence substantially as set out in SEQ ID No:2 or a variant or fragment thereof.

As shown in fig. 1A (2), the first coding sequence may encode an scFv region, which may comprise a VL (variable light chain) sequence and a VH (variable heavy chain) sequence. Preferably, the VL sequence is located upstream (i.e. 5') of the VH sequence. However, in some embodiments, the VH sequence may be located upstream of the VL sequence. Preferably, the VH and VL coding sequences are separated by a linker sequence (e.g., a G4S linker sequence) when in either orientation.

In one embodiment, the VL and VH sequences may be the light chain variable region and the heavy chain variable region of 3G5 (i.e., hybridoma clone name of anti-TCR Vbeta 7.1 monoclonal antibody) for binding to TCR Vbeta 7.1 antigen.

In one embodiment, the first coding sequence (which may encode a VL sequence for binding to the TCR Vbeta, preferably the TCR-Vbeta 7.1 chain) encodes a sequence referred to herein as SEQ ID No:12, as follows:

QVQLQQPGAELVKPGASVKMSCKASGYTFTRYWITWVKQRPGQGLEWIGDIYPGSGFTKYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCAREGGNYWYFDVWGTGTTVTVSS [SEQ ID No:12]

Thus, preferably, the first coding sequence comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:12 or a fragment or variant thereof.

In one embodiment, the first coding sequence (which may encode a VL sequence for binding to a TCR Vbeta, preferably a TCR-Vbeta 7.1 chain) comprises a sequence referred to herein as SEQ ID No:13, as follows:

CAAGTTCAGCTGCAACAGCCTGGCGCCGAGCTTGTGAAACCTGGCGCCTCTGTGAAGATGAGCTGCAAGGCCTCCGGCTACACCTTCACCAGATACTGGATCACCTGGGTCAAGCAGAGGCCTGGACAGGGACTCGAGTGGATCGGCGATATCTATCCTGGCTCCGGCTTCACCAAGTACAACGAGAAGTTCAAGAGCAAGGCCACACTGACCGTGGACACCAGCAGCAGCACAGCCTACATGCAGCTGTCTAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGTGCTAGAGAAGGCGGCAACTACTGGTACTTCGACGTGTGGGGCACCGGCACCACAGTGACAGTTAGTTCT [SEQ ID No:13]

thus, preferably, the first coding sequence comprises a sequence substantially as set forth in SEQ ID No:13 or a fragment or variant thereof.

In one embodiment, the first coding sequence (which may encode a VH sequence for binding a TCR Vbeta, preferably a TCR-Vbeta 7.1 chain) encodes a sequence referred to herein as SEQ ID No:34, as follows:

DIQMTQSPSSLSASLGGKVTLTCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDVATYYCLQYDNLRTFGGGTKLEIKRTD [SEQ ID No:34]

thus, preferably, the first coding sequence comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:34 or a fragment or variant thereof.

In one embodiment, the first coding sequence (which may encode a VH sequence for binding a TCR Vbeta, preferably a TCR-Vbeta 7.1 chain) comprises a sequence referred to herein as SEQ ID No:35, as follows:

GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCTCTCTCGGCGGAAAAGTGACCCTGACATGCAAGGCCAGCCAGGACATCAACAAGTATATCGCCTGGTATCAGCACAAGCCCGGCAAGGGACCTAGACTGCTGATCCACTACACCAGCACACTGCAGCCTGGCATCCCCAGCAGATTTTCTGGCAGCGGCTCCGGCAGAGACTACAGCTTCAGCATCAGCAACCTGGAACCTGAGGACGTGGCCACCTACTACTGCCTGCAGTACGACAACCTGCGGACCTTTGGCGGCGGAACAAAGCTGGAAATCAAGCGGACAGAT [SEQ ID No:35]

thus, preferably, the first coding sequence comprises a sequence substantially as set forth in SEQ ID No:35 or a fragment or variant thereof.

Preferably, the VH (e.g., SEQ ID No: 35) and VL (e.g., SEQ ID No: 13) sequences are separated by a linker sequence when in either orientation. In one embodiment, the linker sequence may be a G4S linker sequence, which may comprise a sequence substantially as set forth in SEQ ID No:10 or a fragment or variant thereof or consists of the amino acid sequence shown in figure 10. Thus, preferably, the linker sequence comprises a sequence substantially as set forth in SEQ ID No:11 or a fragment or variant thereof.

The nucleic acid construct may comprise a nucleotide sequence encoding a CD8a hinge and a Transmembrane (TM) domain. Advantageously, the hinge and TM domains are configured for CAR display and anchoring on CAR-T cells. Preferably, the sequence encoding the hinge and TM domains is located 3' to the first coding sequence.

In one embodiment, the amino acid sequence of the CD8a hinge and transmembrane domain is referred to herein as SEQ ID No:14, as follows:

FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN [SEQ ID No:14]

thus, preferably, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:14 or a fragment or variant thereof.

In one embodiment, the nucleotide sequence encoding the CD8a hinge and transmembrane domain is referred to herein as SEQ ID No:15, as follows:

TTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAAC [SEQ ID No:15]

Thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:15 or a fragment or variant thereof.

The nucleic acid construct may comprise a nucleotide sequence encoding an intracellular domain, which may comprise the signaling domain of CD28, the signaling domain of 4-1BB and/or the CD3 zeta chain, with the signaling domain of CD28, the signaling domain of 4-1BB and the CD3 zeta chain being more preferred. It is understood that these components form the basis of third generation CARs and are necessary to trigger intracellular signaling pathways. Preferably, the intracellular domain is located 3' to the sequence encoding the hinge and transmembrane domains. The signaling domain of CD28 may be 5' to the signaling domain of 4-1 BB. The signaling domain of 4-1BB may be 5' of the CD3 zeta chain.

One embodiment of a CD28 signaling domain may have a sequence referred to herein as SEQ ID No:16, as follows:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[SEQ ID No:16]

thus, preferably, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:16 or a fragment or variant thereof.

In one embodiment, the CD28 signaling domain may be represented by the sequence herein as SEQ ID No:17, as follows:

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGC

CCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

[SEQ ID No:17]

Thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:17 or a fragment or variant thereof.

One embodiment of the 4-1BB signaling domain may have a sequence referred to herein as SEQ ID No:18, as follows:

RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[SEQ ID No:18]

thus, preferably, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:18 or a fragment or variant thereof.

In one embodiment, the 4-1BB signaling domain may be defined herein as SEQ ID No:19, as follows:

CGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAG

AGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[SEQ ID No:19]

thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:19 or a fragment or variant thereof. One embodiment of a CD3 zeta chain may have a sequence referred to herein as SEQ ID No:20, as follows:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG

HDGLYQGLSTATKDTYDALHMQALPPR

[SEQ ID No:20]

thus, preferably, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:20 or a fragment or variant thereof.

In one embodiment, the cd3ζ chain may be defined herein as SEQ ID No:21, as follows:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA

GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG

CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG

CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[SEQ ID No:21]

thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:21 or a fragment or variant thereof.

Preferably, the nucleic acid construct comprises a second coding sequence encoding at least one suicide protein, more preferably at least two suicide proteins. An advantage of the construct of the invention is that a second coding sequence encoding at least one suicide protein is present, which means that the resulting CAR-T cells transduced with the construct can be controllably or inductively detected or eliminated, for example in the event of an adverse reaction in the patient.

Thus, preferably, in one embodiment, provided herein is a nucleic acid construct comprising a promoter operably linked to a first coding sequence encoding an anti-CD 4 Chimeric Antigen Receptor (CAR) and a second coding sequence encoding at least one suicide protein, more preferably at least two suicide proteins.

Preferably, in another embodiment, provided herein is a nucleic acid construct comprising a promoter operably linked to a first coding sequence encoding an anti-T Cell Receptor (TCR) Vbeta CAR and a second coding sequence encoding at least one suicide protein, more preferably at least two suicide proteins.

In yet another embodiment, preferably provided herein is a nucleic acid construct comprising a promoter operably linked to a first coding sequence encoding a plurality of anti-T Cell Receptor (TCR) Vbeta CARs, wherein different Vbeta regions on the T cells are targeted, and a second coding sequence encoding at least one suicide protein, more preferably at least two suicide proteins.

In one embodiment, the second coding sequence may encode an Epidermal Growth Factor Receptor (EGFR) or a truncated epidermal growth factor receptor (tEGFR) (UniProt No. P00533; NCBI reference sequence NP-001333826.1). Expression of tgfr can be controlled by anti-EGFR mAb (cetuximab) to monitor or eliminate CAR-T cells in patients. In one embodiment, the amino acid sequence of tgfr may be referred to herein as SEQ ID No:22, as follows:

MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILK

TVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLF

GTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQA

MNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMV

GALLLLLVVALGIGLFM

[SEQ ID No:22]

thus, preferably, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:22 or a fragment or variant thereof.

In one embodiment, tgfr may consist of the sequence referred to herein as SEQ ID No:23, as follows:

ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAA

TAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGA

TCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAA

ACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAA

TCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCT

CAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTT

GGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGT

GCTCCCCCGAGGGCTGCTGGGGCCCGGAACCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAA

GTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCC

ATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCT

GCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAA

CTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTG

GGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATG

[SEQ ID No:23]

thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:23 or a fragment or variant thereof.

In another embodiment, the second coding sequence may encode an inducible caspase 9 (iC 9). iC9 is a modified human caspase 9 (UniProt No. p55211; NCBI reference sequence np_ 001220.2), fused to human FK506 binding protein (UniProt No. p62942; NCBI reference sequence np_ 000792.1), allowing conditional dimerization using dimerization chemistry inducers (caspase-induced drug (CID), rimiducid), triggering CAR-T cell apoptosis expressing fusion proteins. In one embodiment, the amino acid sequence of iC9 may be referred to herein as SEQ ID No:24, as follows:

MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAY

GATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNID

CEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTS

CPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVDYPYDVPDYALD*

[SEQ ID No:24]

Thus, preferably, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:24 or a fragment or variant thereof.

In one embodiment, iC9 may consist of the amino acid sequence referred to herein as SEQ ID No:25, as follows:

ATGCTCGAGGGAGTGCAGGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACT

ACACCGGGATGCTTGAAGATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGA

GGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTAT

GGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATCTGGCGGTG

GATCCGGAGTCGACGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGA

GCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGAC

TGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGG

CTTTGCTGGAGCTGGCGCAGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCA

CCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGC

TGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCT

CCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCA

GCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGAC

CCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGC

TTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTT

TAAAACATCAGTCGACTATCCGTACGACGTACCAGACTACGCACTCGACTAA

[SEQ ID No:25]

thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:25 or a fragment or variant thereof.

Preferably, the nucleic acid construct of the invention comprises coding sequences encoding EGFR or truncated epidermal growth factor receptor (tgfr) and/or inducible caspase 9 (iC 9). Having one suicide gene can provide powerful control (detection and/or elimination) of CAR and CAR-MAIT cell expression when used in therapy.

More preferably, however, the nucleic acid construct comprises coding sequences encoding a truncated epidermal growth factor receptor (tgfr) and an inducible caspase 9 (iC 9). Advantageously, having two suicide genes can provide tighter control (detection and/or elimination) of CAR and CAR-MAIT cell expression when used in therapy.

Preferably, the nucleic acid construct comprises a nucleotide sequence encoding a peptide spacer, wherein the peptide spacer can be digested (or self-cleaving) to produce the encoded polypeptide as a separate molecule flanking the spacer, e.g., the intracellular domain and the protein encoded by the suicide gene, which can be tgfr and/or iC9. Thus, a peptide spacer may be referred to as a self-cleaving peptide.

Preferably, the spacer sequence comprises and encodes a viral peptide spacer sequence, more preferably a viral 2A peptide spacer sequence (Furler S, paterna J-C, weibel M and Bueler H Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons Gene Ther.2001,vol.8,PP:864-873).

Preferably, the spacer sequence encoding the 2A peptide sequence links the sequence encoding the intracellular domain to the sequence encoding the suicide protein. In embodiments where the construct encodes two suicide proteins (e.g., EGFR/tgfr and iC 9), the nucleic acid construct comprises a first spacer sequence disposed between the sequence encoding the intracellular domain and the sequence encoding the first suicide protein, and a second spacer sequence disposed between the sequence encoding the first suicide protein and the sequence encoding the second suicide protein. The first suicide protein may be EGFR/tgfr and the second suicide protein may be iC9. In another embodiment, the first suicide protein may be iC9 and the second suicide protein may be EGFR/tgfr.

The 2A spacer sequence may be any known variant, including those sequences known as E2A, F2A, P A and T2A, as described in Wang Y et al (Wang Y et al scientific Reports 2015,5). Preferably, the self-cleaving peptide is P2A. In one embodiment, the P2A spacer has a sequence referred to herein as SEQ ID No:26, as follows:

GSGATNFSLLKQAGDVEENPGP

[SEQ ID No:26]

Thus, preferably, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:26 or a fragment or variant thereof.

In one embodiment, the 2A spacer may consist of the sequence referred to herein as SEQ ID No:27, as follows:

GGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCT

thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:27 or a fragment or variant thereof.

In further embodiments, the construct may further comprise a nucleotide sequence encoding a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) that enhances expression of the transgene. Preferably, the WPRE coding sequence is located 3' to the suicide protein coding sequence. In particular, the WPRE sequence is preferably 3' of the iC9 coding sequence.

One embodiment of the WPRE is 592bp long, comprising a γ - α - β element, and is referred to herein as SEQ ID No:28, as follows:

AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTG

CTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTA

TGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCC

ACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT

GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTG

TGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTG

CTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

[SEQ ID NO:28]

preferably, the nucleic acid comprises a nucleotide sequence substantially as set forth in SEQ ID No:28 or a fragment or variant thereof.

Preferably, the construct comprises left (i.e., 5 ') and/or right (i.e., 3') Long Terminal Repeats (LTRs). Preferably, each LTR is located at the 5 'and/or 3' end of the construct.

In a preferred embodiment, the nucleic acid construct comprises a 5' promoter in this particular order; a sequence encoding an scFv specific for the CD4 or TCR Vbeta region; and a 3' sequence encoding an intracellular domain. The use of 5 'and 3' indicates that the feature is upstream or downstream and is not intended to indicate that the feature is necessarily a terminal feature.

In a preferred embodiment, the nucleic acid construct comprises a 5' promoter in this particular order; a sequence encoding an scFv specific for the CD4 or TCR Vbeeta region; a sequence encoding an intracellular domain; and a 3' sequence encoding at least one suicide protein.

In a more preferred embodiment, the nucleic acid construct comprises a 5' promoter (preferably PGK promoter) in this particular order; a sequence encoding an scFv specific for CD4 or one or more TCR Vbeta regions (preferably VL and/or VH domains); sequences encoding intracellular domains (preferably CD28, 4-1BB and/or CD3 zeta chains); 3' sequences encoding at least one suicide protein, preferably EGFR/EGFRt and/or iC 9.

In yet a more preferred embodiment, the nucleic acid construct comprises a 5' promoter (preferably PGK promoter) in this particular order; a sequence encoding a Signal Peptide (SP); a sequence encoding an scFv specific for CD4 or one or more TCR Vbeta regions (VL and VH domains preferably separated by a G4S linker); sequences encoding a CD8a hinge and a transmembrane domain; sequences encoding intracellular domains (preferably CD28, 4-1BB and CD3 zeta chains); the 3' sequence encoding at least one suicide protein (preferably EGFR/EGFR and iC 9), optionally with a self-cleaving peptide spacer between the sequence encoding the intracellular domain and the suicide protein coding sequence.

In a first most preferred embodiment (referred to as "CART 4"), the nucleic acid construct comprises the 5' pgk promoter in this particular order; a sequence encoding a Signal Peptide (SP); sequences encoding VL and VH domains of scFv specific for CD4 (preferably separated by a G4S linker); sequences encoding a CD8a hinge and a transmembrane domain; sequences encoding the CD28, 4-1BB and CD3 zeta chains of the intracellular domain; a sequence encoding a first self-cleaving peptide spacer; a sequence encoding EGFR/EGFRt; a sequence encoding a second self-cleaving peptide spacer; 3' sequence encoding iC 9.

In a second most preferred embodiment (referred to as "cartvb7.1"), the nucleic acid construct comprises the 5' pgk promoter in this particular order; a sequence encoding a Signal Peptide (SP); sequences encoding VL and VH domains of scFv specific for one or more TCR Vbeta regions, preferably TCR-Vbeta 7.1 chains (preferably separated by a G4S linker); sequences encoding a CD8a hinge and a transmembrane domain; sequences encoding the CD28, 4-1BB and CD3 zeta chains of the intracellular domain; a sequence encoding a first self-cleaving peptide spacer; a sequence encoding EGFR/EGFRt; a sequence encoding a second self-cleaving peptide spacer; 3' sequence encoding iC 9.

Thus, a preferred embodiment of the nucleic acid construct (referred to as "CART 4") has a sequence referred to herein as SEQ ID No:29, as follows:

METDTLLLWVLLLWVPGSTGDDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGV

PDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSC

KASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGA

WFAYWGQGTLVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG

VLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK

MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPA

FLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPEN

RTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENS

CKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCA

HYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

GSGATNFSLLKQAGDVEENPGPMLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWE

EGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCL

IINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGA

VYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISS

LPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVDYPYDVPDYALD*

[SEQ ID No:29]

Thus, preferably, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:29 or a fragment or variant thereof.

Preferably, an embodiment of the nucleic acid construct (referred to as "CART 4") has a sequence referred to herein as SEQ ID No:30, as follows:

ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGACGACATTGTGATGACTCAGAGCC

CCGACAGCCTGGCCGTCTCACTGGGCGAAAGGGTGACCATGAATTGTAAATCTTCTCAGAGCCTGCTGTACAGTACAAACCAGAA

AAATTACCTGGCCTGGTATCAGCAGAAACCCGGCCAGAGCCCTAAGCTGCTGATCTATTGGGCAAGTACCCGAGAGTCAGGAGTG

CCAGACAGATTCTCCGGGTCTGGAAGTGGCACAGACTTCACCCTGACAATTAGCTCCGTGCAGGCCGAGGACGTGGCTGTCTACT

ATTGCCAGCAGTACTATAGCTACCGAACTTTCGGCGGGGGAACCAAACTGGAAATCAAGGGAGGAGGAGGCAGTGGCGGAGGAGG

GTCAGGAGGAGGAGGAAGCCAGGTGCAGCTGCAGCAGTCCGGACCAGAGGTGGTCAAACCCGGCGCTAGCGTCAAAATGTCCTGT

AAGGCATCTGGCTACACTTTCACCTCTTATGTGATTCACTGGGTCAGACAGAAGCCTGGGCAGGGACTGGACTGGATCGGGTACA

TTAACCCATATAATGATGGAACTGACTACGATGAAAAGTTTAAAGGCAAGGCCACACTGACTTCCGACACCTCAACAAGCACTGC

TTATATGGAGCTGTCTAGTCTGAGGTCTGAAGACACAGCAGTGTACTATTGCGCCCGCGAGAAGGATAACTACGCCACTGGCGCT

TGGTTTGCATATTGGGGCCAGGGGACCCTGGTGACAGTCTCATCCGCGGCCGCATTCGTGCCGGTCTTCCTGCCAGCGAAGCCCA

CCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCC

AGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGG

GTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACA

TGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC

CCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAA

GAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACG

CCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAG

ACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAG

ATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA

CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGATCCGGAGCCACGAACTTCTCTCTGTTAAA

GCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCA

TTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTA

AACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCC

TCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAAC

AGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCA

GCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTG

CTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGC

TGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAACCCAGGGACTGCGTCTCTTGCC

GGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTG

CATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCC

CACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAG

ACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGG

GCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATG

GGATCTGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGCTCGAGGGAGTGCAGG

TGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGA

TGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAA

GAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAG

GCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGATT

TGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTC

ATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTC

GCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCA

GCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCT

GTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGA

AGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGA

GTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGT

TTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGT

ACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGT

TTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGTCGACTAT

CCGTACGACGTACCAGACTACGCACTCGACTAA

[SEQ ID No:30]

thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:30 or a fragment or variant thereof.

Thus, a second preferred embodiment of the nucleic acid construct (referred to as "cartvb7.1") has a sequence referred to herein as SEQ id no:31, as follows:

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASLGGKVTLTCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSG

SGSGRDYSFSISNLEPEDVATYYCLQYDNLRTFGGGTKLEIKRTDGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKAS

GYTFTRYWITWVKQRPGQGLEWIGDIYPGSGFTKYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCAREGGNYWYFDVWG

TGTTVTVSSAAAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL

SLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDG

CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA

YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMLLLVTSLLLCELPHPAFLLI

PRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDL

HAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKAT

GQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID

GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMGSGA

TNFSLLKQAGDVEENPGPMLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVA

QMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINN

VNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGT

DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTP

SDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVDYPYDVPDYALD*

[SEQ ID No:31]

preferably, therefore, the construct comprises a sequence encoding a sequence substantially as set forth in SEQ ID No:31 or a fragment or variant thereof.

Preferably, an embodiment of the nucleic acid construct (referred to as "cartvb7.1") has a sequence referred to herein as SEQ ID No:32, as follows:

ATGGCTCTGCCTGTTACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCTGACATCCAGATGACACAGAGCC

CTAGCAGCCTGTCTGCCTCTCTCGGCGGAAAAGTGACCCTGACATGCAAGGCCAGCCAGGACATCAACAAGTATATCGCCTGGTA

TCAGCACAAGCCCGGCAAGGGACCTAGACTGCTGATCCACTACACCAGCACACTGCAGCCTGGCATCCCCAGCAGATTTTCTGGC

AGCGGCTCCGGCAGAGACTACAGCTTCAGCATCAGCAACCTGGAACCTGAGGACGTGGCCACCTACTACTGCCTGCAGTACGACA

ACCTGCGGACCTTTGGCGGCGGAACAAAGCTGGAAATCAAGCGGACAGATGGCGGAGGCGGATCAGGCGGCGGAGGAAGCGGTGG

CGGAGGATCTCAAGTTCAGCTGCAACAGCCTGGCGCCGAGCTTGTGAAACCTGGCGCCTCTGTGAAGATGAGCTGCAAGGCCTCC

GGCTACACCTTCACCAGATACTGGATCACCTGGGTCAAGCAGAGGCCTGGACAGGGACTCGAGTGGATCGGCGATATCTATCCTG

GCTCCGGCTTCACCAAGTACAACGAGAAGTTCAAGAGCAAGGCCACACTGACCGTGGACACCAGCAGCAGCACAGCCTACATGCA

GCTGTCTAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGTGCTAGAGAAGGCGGCAACTACTGGTACTTCGACGTGTGGGGC

ACCGGCACCACAGTGACAGTTAGTTCTGCGGCCGCGGCCGCATTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAG

CGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGG

CGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTG

TCACTGGTTATCACCCTTTACTGCAACCACAGGAACAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTC

CCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCGTTTCTCTGT

TGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGC

TGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACC

AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGA

CCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCC

TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGG

ACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGA

CGTGGAAGAAAACCCCGGTCCTATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATC

CCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAA

ACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCC

ACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTC

CATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAA

CATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATAC

AATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACA

GGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAACCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCC

GAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCA

CCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGAC

GGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATG

TGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCC

GTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGGGATCTGGAGCC

ACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGCTCGAGGGAGTGCAGGTGGAAACCATCT

CCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGT

TGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTGCC

CAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCAC

CACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGATTTGGTGATGTCGG

TGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAAT

GTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGC

TGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGGACCACGG

TGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACA

GATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCT

TTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAG

TAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCC

AGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCC

TGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGG

GATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGTCGACTATCCGTACGACGTA

CCAGACTACGCACTCGACTAA

[SEQ ID No:32]

thus, preferably, the construct comprises a sequence substantially as set forth in SEQ ID No:32 or a fragment or variant thereof.

Thus, it will be appreciated that the isolated MAIT cell obtained using the method of the second or third aspect may be activated and eventually transduced with a nucleic acid construct encoding a CAR according to the fifth aspect, thereby producing a CAR-MAIT cell of the first or fourth aspect.

In a sixth aspect, provided herein is an expression vector encoding the nucleic acid construct of the fifth aspect.

Preferably, the vector is recombinant. Preferably, the vector is a viral vector, more preferably a retroviral vector. Figures 9 and 10 show maps of features of two preferred embodiments of the vectors of the present invention.

In one embodiment (CART 4: CAR4-tEGFR-iC9; see FIG. 9), the vector has a sequence designated herein as SEQ ID No:33, as follows:

TGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAG

AAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTC

AGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAG

GACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTC

AATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTATTCCCAATAAAGCC

TCTTGCTGTTTGCATCCGAATCGTGGACTCGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACCTCGGGGGTCTT

TCATTTGGAGGTTCCACCGAGATTTGGAGACCCCTGCCCAGGGACCACCGACCCCCCCGCCGGGAGGTAAGCTGGCCAGCGGTCG

TTTCGTGTCTGTCTCTGTCTTTGTGCGTGTTTGTGCCGGCATCTAATGTTTGCGCCTGCGTCTGTACTAGTTAGCTAACTAGCTC

TGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCTGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTTGGGGGCC

GTTTTTGTGGCCCGACCTGAGGAAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATGTGGTTCTGGTAGGAGACGAGAACCT

AAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGAACCGAAGCCGCGCGTCTTGTCTGCTGCAGCGCTGCAGCATC

GTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCT

TAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGC

AGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT

TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCA

AGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCC

TCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCGGAATTAGATCTCTCGAGGTTAACGAATTCTACCGGGTAGGG

GAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTC

GCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGT

CAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATG

GACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGA

GGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGC

ATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATTCTCCGGGCCTTTCGACCTGCAGCCCAAGCCA

CCATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGACGACATTGTGATGACTCAGAG

CCCCGACAGCCTGGCCGTCTCACTGGGCGAAAGGGTGACCATGAATTGTAAATCTTCTCAGAGCCTGCTGTACAGTACAAACCAG

AAAAATTACCTGGCCTGGTATCAGCAGAAACCCGGCCAGAGCCCTAAGCTGCTGATCTATTGGGCAAGTACCCGAGAGTCAGGAG

TGCCAGACAGATTCTCCGGGTCTGGAAGTGGCACAGACTTCACCCTGACAATTAGCTCCGTGCAGGCCGAGGACGTGGCTGTCTA

CTATTGCCAGCAGTACTATAGCTACCGAACTTTCGGCGGGGGAACCAAACTGGAAATCAAGGGAGGAGGAGGCAGTGGCGGAGGA

GGGTCAGGAGGAGGAGGAAGCCAGGTGCAGCTGCAGCAGTCCGGACCAGAGGTGGTCAAACCCGGCGCTAGCGTCAAAATGTCCT

GTAAGGCATCTGGCTACACTTTCACCTCTTATGTGATTCACTGGGTCAGACAGAAGCCTGGGCAGGGACTGGACTGGATCGGGTA

CATTAACCCATATAATGATGGAACTGACTACGATGAAAAGTTTAAAGGCAAGGCCACACTGACTTCCGACACCTCAACAAGCACT

GCTTATATGGAGCTGTCTAGTCTGAGGTCTGAAGACACAGCAGTGTACTATTGCGCCCGCGAGAAGGATAACTACGCCACTGGCG

CTTGGTTTGCATATTGGGGCCAGGGGACCCTGGTGACAGTCTCATCCGCGGCCGCATTCGTGCCGGTCTTCCTGCCAGCGAAGCC

CACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGG

CCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTG

GGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTA

CATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGC

TCCCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTC

AAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGA

CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG

AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA

AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAG

TACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGATCCGGAGCCACGAACTTCTCTCTGTTA

AAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAG

CATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATAT

TAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACT

CCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAA

ACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGT

CAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTG

TGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACA

GCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAACCCAGGGACTGCGTCTCTTG

CCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAG

TGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTG

CCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGC

AGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAAT

GGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCA

TGGGATCTGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGCTCGAGGGAGTGCA

GGTGGAAACCATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAA

GATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGG

AAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCC

AGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGA

TTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCC

TCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCG

TCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCG

CAGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGG

CTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGG

GAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGAC

GAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTA

GTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTG

GTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCT

GTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGTCGACT

ATCCGTACGACGTACCAGACTACGCACTCGACTAAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTT

AACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTT

TCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGT

GTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATT

GCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT

CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTC

GGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACG

AGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTATCGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAA

GACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTT

CAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGC

CAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCT

GAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAA

AAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTG

CAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATG

GGTAACAGTTTCTTGAAGTTGGAGAACAACATTCTGAGGGTAGGAGTCGAATATTAAGTAATCCTGACTCAATTAGCCACTGTTT

TGAATCCACATACTCCAATACTCCTGAAATAGTTCATTATGGACAGCGCAGAAGAGCTGGGGAGAATTAATTCGTAATCATGGTC

ATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGT

GCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATT

AATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT

CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC

ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG

AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTC

CCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAT

AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC

GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG

GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTT

GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG

GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA

CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA

AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT

CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGG

CCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG

CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTA

ATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTC

CCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT

AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTG

TGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAA

TACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTG

TTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA

AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA

TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGC

ACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGC

CCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGC

GGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAG

CAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGC

CATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCGCAAGGAATGGTGCATGCAAGGAGA

TGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCG

ATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCG

TAGAGGCGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGC

CTATAGAGTACGAGCCATAGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAA

[SEQ ID NO:33]

preferably, the vector comprises a sequence substantially as set forth in SEQ ID NO:33 or a fragment or variant thereof.

In one embodiment (CARTVb7.1: CARTVb7.1-tEGFR-iC9; see FIG. 10), the vector has a sequence referred to herein as SEQ ID No:36, as follows:

TGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAG

AAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTC

AGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAG

GACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTC

AATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTATTCCCAATAAAGCC

TCTTGCTGTTTGCATCCGAATCGTGGACTCGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACCTCGGGGGTCTT

TCATTTGGAGGTTCCACCGAGATTTGGAGACCCCTGCCCAGGGACCACCGACCCCCCCGCCGGGAGGTAAGCTGGCCAGCGGTCG

TTTCGTGTCTGTCTCTGTCTTTGTGCGTGTTTGTGCCGGCATCTAATGTTTGCGCCTGCGTCTGTACTAGTTAGCTAACTAGCTC

TGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCTGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTTGGGGGCC

GTTTTTGTGGCCCGACCTGAGGAAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATGTGGTTCTGGTAGGAGACGAGAACCT

AAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGAACCGAAGCCGCGCGTCTTGTCTGCTGCAGCGCTGCAGCATC

GTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCT

TAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGC

AGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTT

TCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCA

AGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCC

TCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCGGAATTAGATCTCTCGAGGTTAACGAATTCTACCGGGTAGGG

GAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTC

GCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGT

CAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATG

GACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGA

GGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGC

ATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATTCTCCGGGCCTTTCGACCTGCAGCCCAAGCCA

CCATGGCTCTGCCTGTTACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCAGACCTGACATCCAGATGACACAGAG

CCCTAGCAGCCTGTCTGCCTCTCTCGGCGGAAAAGTGACCCTGACATGCAAGGCCAGCCAGGACATCAACAAGTATATCGCCTGG

TATCAGCACAAGCCCGGCAAGGGACCTAGACTGCTGATCCACTACACCAGCACACTGCAGCCTGGCATCCCCAGCAGATTTTCTG

GCAGCGGCTCCGGCAGAGACTACAGCTTCAGCATCAGCAACCTGGAACCTGAGGACGTGGCCACCTACTACTGCCTGCAGTACGA

CAACCTGCGGACCTTTGGCGGCGGAACAAAGCTGGAAATCAAGCGGACAGATGGCGGAGGCGGATCAGGCGGCGGAGGAAGCGGT

GGCGGAGGATCTCAAGTTCAGCTGCAACAGCCTGGCGCCGAGCTTGTGAAACCTGGCGCCTCTGTGAAGATGAGCTGCAAGGCCT

CCGGCTACACCTTCACCAGATACTGGATCACCTGGGTCAAGCAGAGGCCTGGACAGGGACTCGAGTGGATCGGCGATATCTATCC

TGGCTCCGGCTTCACCAAGTACAACGAGAAGTTCAAGAGCAAGGCCACACTGACCGTGGACACCAGCAGCAGCACAGCCTACATG

CAGCTGTCTAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGTGCTAGAGAAGGCGGCAACTACTGGTACTTCGACGTGTGGG

GCACCGGCACCACAGTGACAGTTAGTTCTGCGGCCGCGGCCGCATTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCC

AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGG

GGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCC

TGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGAC

TCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCCGTTTCTCT

GTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG

GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA

CCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGG

GACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGG

CCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA

GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGA

GACGTGGAAGAAAACCCCGGTCCTATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGA

TCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAA

AAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGAT

CCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACC

TCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACAT

AACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAAT

ACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCA

CAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAACCCAGGGACTGCGTCTCTTGCCGGAATGTCAG

CCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGC

CACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTG

ACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCA

TGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATC

CCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGGGATCTGGAG

CCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGCTCGAGGGAGTGCAGGTGGAAACCAT

CTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAA

GTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAAGAAGGGGTTG

CCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCC

ACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATCTGGCGGTGGATCCGGAGTCGACGGATTTGGTGATGTC

GGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACA

ATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTC

GCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGGACCAC

GGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCA

CAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCT

CTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGC

AGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACAC

CCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGAC

CCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAA

GGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGTCGACTATCCGTACGACG

TACCAGACTACGCACTCGACTAAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCT

CCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGT

ATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGC

AACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAA

CTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCAT

CGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCC

AGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCC

CTTTGGGCCGCCTCCCCGCCTATCGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGT

AGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTT

AGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATG

GTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTG

TGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAA

CCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCG

ACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATGGGTAACAGTTTC

TTGAAGTTGGAGAACAACATTCTGAGGGTAGGAGTCGAATATTAAGTAATCCTGACTCAATTAGCCACTGTTTTGAATCCACATA

CTCCAATACTCCTGAAATAGTTCATTATGGACAGCGCAGAAGAGCTGGGGAGAATTAATTCGTAATCATGGTCATAGCTGTTTCC

TGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTG

AGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCC

AACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCG

GCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA

GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAA

ATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTC

TCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGT

AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTAT

CCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGC

GAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCT

CTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTG

TTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAA

CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTT

AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTC

TATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGC

AATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT

CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA

ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG

GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA

GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT

ACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACA

TAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGT

TCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGC

AAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTA

TCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA

AAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCG

CGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAG

CAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTG

AGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGC

GCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAG

TTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACA

GTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATC

GGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGCGATTA

GTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACG

AGCCATAGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAA

[SEQ ID NO:36]

preferably, the vector comprises a sequence substantially as set forth in SEQ ID NO:36 or a fragment or variant thereof.

Thus, it will be appreciated that the isolated MAIT cell obtained using the method of the second or third aspect may be activated and eventually transduced with a CAR-encoding expression vector according to the sixth aspect to produce a CAR-MAIT cell of the first or fourth aspect.

In an eleventh aspect, provided herein is a T cell comprising a construct according to the fifth construct or a vector according to the sixth aspect, optionally wherein the T cell expresses an anti-CD 4 Chimeric Antigen Receptor (CAR) or an anti-Vbeta CAR.

Preferably, the T cells are mucosa-associated constant T (MAIT) cells.

In a seventh aspect, provided herein is a pharmaceutical composition comprising T cells according to the eleventh aspect, preferably MAIT cells according to the first or fourth aspect, and a pharmaceutically acceptable excipient.

Preferably, the pharmaceutical composition comprises a plurality of T cells or MAIT cells of the present invention. For example, the composition may comprise at least 100, 1000, or 10000T cells or MAIT cells. Preferably, the composition comprises at least 100000, at least 1000000 or at least 10000000T cells or MAIT cells.

In an eighth aspect, provided herein is a T cell according to the eleventh aspect, a MAIT cell according to the first or fourth aspect or a pharmaceutical composition of the seventh aspect for use in therapy.

In a ninth aspect, provided herein is a pharmaceutical composition according to the seventh aspect of a T cell according to the eleventh aspect, a MAIT cell according to the first or fourth aspect for use in (i) immunotherapy; (ii) treating, preventing or ameliorating cancer; (ii) treating, preventing or ameliorating a microbial infection; or (iv) treating, preventing or ameliorating an autoimmune disease.

In a tenth aspect, the present invention provides a method of: (i) Treating, preventing or ameliorating a disease in a subject with immunotherapy; (ii) treating, preventing or ameliorating cancer; (iii) Treating, preventing or ameliorating a microbial infection in a subject; or (iv) treating, preventing or ameliorating an autoimmune disease in a subject, the method comprising administering to a patient in need of such treatment or having administered a therapeutically effective amount of T cells according to the eleventh aspect, MAIT cells according to the first or fourth aspects or a pharmaceutical composition of the seventh aspect.

Preferably, the T cells, the MAIT cells or the pharmaceutical composition are used for the treatment, prevention or amelioration of T cell malignancies, which may be solid tumors or liquid tumors.

The T cell malignancy may be Peripheral T Cell Lymphoma (PTCL) or Cutaneous T Cell Lymphoma (CTCL).

Peripheral T Cell Lymphomas (PTCLs) include a number of rare invasive diseases in which the T cells of patients become cancerous. PTCL is divided into three classes, i.e., intranodal, extranodal and leukemia, each of which is encompassed by the present invention.

The PTCL may be a PTCL subtype selected from the group consisting of: adult T cell acute lymphocytic lymphoma or leukemia (ATL), enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous lipomyotic lymphoma (SPTCL), precursor T cell acute lymphocytic lymphoma or leukemia, and angioimmunoblastic T cell lymphoma (AITL).

Adult T cell acute lymphoblastic lymphoma or leukemia (ATL) is more common in japan and the caribbean region than in the united states and is associated with human T cell leukemia virus-1 (HTLV-1). Intestinal disease-associated lymphomas are associated with celiac disease, a chronic intestinal disease, caused by allergies to gluten proteins in wheat, rye and barley. Symptoms typically include stomach pain, weight loss, gastrointestinal bleeding, or intestinal perforation. Treatment of patients with enteropathy-associated T-cell lymphomas includes anthracycline-based chemotherapy regimens, nutritional supplements, and (if applicable) gluten-free diets. Hepatosplenic lymphoma is an extremely rare invasive disease, beginning in the liver or spleen, and commonly affecting young people over 20 and 30 years of age. Treatment of patients with hepatosplenic T cell lymphomas includes anthracycline-based chemotherapy and in some cases stem cell transplantation.

Subcutaneous lipoteulitis-like lymphoma (SPTCL) is the least rare and well-defined T cell lymphoma. This lymphoma occurs mainly in subcutaneous adipose tissue, leading to nodule formation. Symptoms include fever, chills, weight loss, and ulceration of the oral mucosa. SPTCL may be rapidly aggressive or inert (slow growing). Treatment includes anthracycline-based combination chemotherapy or local radiotherapy. Precursor T cell acute lymphocytic lymphomas or leukemias may be diagnosed as leukemia or lymphoma or both. This cancer is found in both children and adults, most commonly in adolescents and adult males. The treatment of patients newly diagnosed with precursor T cell acute lymphoblastic lymphoma or leukemia is active chemotherapy and radiation therapy. NelarabineIs approved for the treatment of recurrent or refractory precursor T cell acute lymphocytic lymphomas or leukemias in adults and children.

Angioimmunoblastic T cell lymphoma (AITL) is a tumor characterized by strong inflammatory and immune responses, which is evidenced by clinical, pathological, cellular and biological properties. Since tumor cells are phenotypically similar to follicular helper T (Tfh) cells, they are considered to function somewhat similar to non-neoplastic Tfh cells seen in reactive follicular hyperplasia. However, in most AITL cases, the follicles do not proliferate, but rather are depleted or destroyed. It has recently been reported that AITL comprises 36.1% of PTCL.

Cutaneous T Cell Lymphoma (CTCL) accounts for about 70-75% of primary cutaneous lymphomas. CTCL may be a CTCL subtype selected from the group consisting of: mycosis Fungoides (MF), sezary Syndrome (SS), and cd4+ small and medium polymorphic T cell lymphoproliferative diseases.

Mycosis Fungoides (MF) is the most common subtype. Cerclari syndrome (SS) is a more aggressive CTCL type. SS patients suffer from erythroderma (i.e. rash affecting >80% of body surface area BSA), lymphadenopathy, and the presence of large numbers of circulating neoplastic cd4+ T cells in peripheral blood.

In other embodiments, T cells, MAIT cells, or pharmaceutical compositions may be used to treat, prevent, or ameliorate a viral (e.g., HIV, HBV, HTLV, EBV, HPV), bacterial (e.g., TB), or fungal infection.

In other embodiments, T cells, MAIT cells, or pharmaceutical compositions can be used to treat, prevent, or ameliorate autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, or myasthenia gravis.

Preferably, the method comprises triggering a sequence encoding a suicide protein. Thus, in embodiments where the nucleic acid construct comprises a sequence encoding EGFR or tgfr, the method preferably comprises administering an anti-EGFR antibody to the subject. For example, the anti-EGFR antibody may be the monoclonal antibody cetuximab. Administration of the antibody is capable of monitoring or eliminating CAR-T cells in the subject.

In embodiments where the nucleic acid construct comprises a sequence encoding iC9, the method may comprise administering a caspase-inducing drug (CID) to the subject. For example, the CID may include Rimiducid. Administration of CID can conditionally dimerize caspases, triggering apoptosis of CAR-T cells expressing the fusion protein, resulting in depletion of CAR-T cells in the subject.

In a most preferred embodiment, the construct encodes two suicide proteins, including iC9 and tgfr, so the use of an anti-EGFR antibody and CID can precisely control the longevity of CAR-T or CAR-MAIT cells in a subject receiving treatment.

It will be appreciated that CAR-T cells or CAR-MAIT cells (collectively referred to herein as "agents") according to the present invention can be used in monotherapy (e.g. T cells or CAR-MAIT cells alone) for therapy (preferably in immunotherapy), wherein (i) the treatment, amelioration or prevention of cancer or T cell malignancy; or (ii) treating, preventing or ameliorating a microbial infection, or (iii) an autoimmune disease. Alternatively, the CAR-T or CAR-MAIT cells according to the invention may be used as an adjunct to or in combination with known immunotherapy, or for the treatment of microbial infections as well as cancer or autoimmune diseases.

The agents of the invention may be combined in compositions having a variety of different forms, depending on the manner of use of the composition. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposome suspension, or any other form suitable for administration to a human or animal in need thereof. It will be appreciated that the pharmaceutical carrier according to the invention should be a carrier that is well tolerated by the subject to whom it is administered.

Agents comprising the agents of the invention may be used in a variety of ways. For example, oral administration may be desired, in which case the agent may be contained within a composition that may be orally ingested, for example, in the form of a tablet, capsule, or liquid. The compositions comprising the agents and medicaments of the present invention may be administered by inhalation (e.g., intranasally). The compositions may also be formulated for topical use. For example, a cream or ointment may be applied to the skin.

The agents and medicaments according to the invention may also be incorporated into slow-release or delayed-release devices. Such devices may be inserted, for example, on or under the skin, and the drug may be released over weeks or even months. The device may be positioned at least adjacent to the treatment site. Such devices may be particularly advantageous when long-term treatment with the agents according to the invention is required and frequent administration (e.g. at least daily injection) is often required.

In a preferred embodiment, the agents and medicaments according to the invention can be administered to a subject by injection into the blood stream or directly into a site in need of treatment. The injection may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion) or intradermal (bolus or infusion).

It will be appreciated that the amount of genetic construct or vector (i.e., agent) required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, physicochemical properties of the agent, and whether it is used as monotherapy or in combination therapy. The frequency of administration will also be affected by the half-life of the agent in the subject being treated. The optimal dosage to be administered can be determined by one of skill in the art and will vary with the particular agent used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the disease being treated (e.g., cancer, T cell malignancy, microbial infection, or autoimmune disease). Other factors depending on the particular subject being treated will result in the need to adjust dosages, including subject age, weight, sex, diet, and time of administration.

In general, daily doses of the agent of the invention of between 0.001 μg/kg body weight and 10mg/kg body weight can be used for therapy, in particular for the treatment, amelioration or prophylaxis of cancer, T cell malignancy, microbial infection or autoimmune disease, depending on the particular agent. More preferably, the daily dose of agent is from 0.01 μg/kg body weight to 1mg/kg body weight, more preferably from 0.1 μg/kg to 100 μg/kg body weight, and most preferably from about 0.1 μg/kg to 10 μg/kg body weight.

Alternatively, the dose administered to the subject may be at 0.5x10 ⁷ To 5x10 ¹² Transduction Units (TU)/Kg body weight. More preferably, the dose administered to the subject may be at 0.5x10 ⁸ To 5x10 ¹¹ TU/Kg body weight. Most preferably, the dose administered to the subject may be at 0.5x10 ⁹ To 5x10 ¹⁰ TU/Kg body weight.

The agent may be administered before, during or after the onset of cancer, T cell malignancy, microbial infection or autoimmune disease. Daily doses may be administered as a single administration (e.g., a single daily injection). Alternatively, the agent may require two or more administrations per day. As an example, the agent may be administered at a dose of between 0.07 μg and 700mg (i.e. assuming a weight of 70 kg) twice daily (or more times depending on the severity of the disease being treated, e.g. cancer). The patient receiving treatment may be administered a first dose at wake-up and then a second dose at night (if a two dose regimen is employed) or every 3 or 4 hours thereafter. Alternatively, the agent may need to be administered once a week, or even once a month. Alternatively, a sustained release device may be used to provide the patient with an optimal dose of the agent according to the invention without the need to administer repeated doses. Known procedures, such as those conventionally employed in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), may be used to form specific formulations of agents according to the invention and precise treatment regimens (e.g., daily doses and dosing frequency of the agents).

The pharmaceutical composition of the present invention is preferably an immunotherapeutic therapeutic composition, an autoimmune disease therapeutic composition, an anti-infective composition or an anti-cancer composition, i.e. a pharmaceutical formulation for therapeutic amelioration, prevention or treatment of cancer in a subject.

In an eleventh aspect, the invention also provides a method of preparing a pharmaceutical composition according to the seventh aspect, the method comprising combining a therapeutically effective amount of a MAIT cell according to the first or fourth aspect with a pharmaceutically acceptable carrier.

The "subject" may be a vertebrate, mammal, or livestock. Thus, the medicament according to the invention may be used for the treatment of any mammal, such as livestock (e.g. horses), pets, or may be used for other veterinary applications. Most preferably, the subject is a human.

A "therapeutically effective amount" of a genetic construct or vector is any amount of an agent that is required to treat a disease (e.g., cancer) or produce a desired effect when administered to a subject.

For example, a therapeutically effective amount of the genetic construct or vector used may be from about 0.001ng to about 1mg, preferably from about 0.01ng to about 100ng. Preferably, the amount of genetic construct or vector is from about 0.1ng to about 10ng, most preferably from about 0.5ng to about 5ng.

Reference herein to a "pharmaceutically acceptable carrier" is to any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

In one embodiment, the pharmaceutically acceptable carrier may be a solid and the composition may be in the form of a powder or tablet. The solid pharmaceutically acceptable carrier may include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings or tablet disintegrating agents. The carrier may also be an encapsulating material. In powders, the carrier is a finely divided solid which is admixed with the finely divided active agent according to the invention. In tablets, the active agent may be mixed with a carrier having the necessary compression characteristics in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% active agent. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical carrier may be a gel and the composition may be in the form of a cream or the like.

However, the pharmaceutical carrier may be a liquid and the pharmaceutical composition in the form of a solution. Liquid carriers can be used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agents of the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid carrier may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colorants, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as described above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., ethylene glycol) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers can be used in sterile liquid form compositions for parenteral administration. The liquid carrier for the pressurized composition may be a halocarbon or other pharmaceutically acceptable propellant.

The liquid pharmaceutical composition is a sterile solution or suspension and can be used by intramuscular injection, intrathecal injection, epidural injection, intraperitoneal injection, intravenous injection, and in particular subcutaneous injection. The agent may be formulated as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline or other suitable sterile injectable medium.

The agents and compositions of the invention may be administered orally in the form of sterile solutions or suspensions containing other solutes or suspensions (e.g., sufficient saline or dextrose to render the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid esters of sorbitol and its anhydrides copolymerized with ethylene oxide), and the like. The agents used according to the invention may also be administered orally in the form of liquid or solid compositions. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets and powders, as well as liquid forms such as solutions, syrups, elixirs and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions and suspensions.

It is to be understood that the invention extends to any nucleic acid or peptide, or variant, derivative or analogue thereof, comprising essentially an amino acid or nucleic acid sequence of any of the sequences mentioned herein, including variants or fragments thereof. The terms "substantially amino acid/nucleotide/peptide sequence", "variant" and "fragment" may be sequences having at least 40% sequence identity to an amino acid/nucleotide/peptide sequence of any of the sequences mentioned herein. For example, 40% identity with the sequences identified by SEQ ID No. 1 to SEQ ID No. 36, etc.

Amino acid/polynucleotide/polypeptide sequences having a sequence identity of greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, more preferably greater than 80% to any of the sequences described above are also contemplated. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, most preferably at least 99% identity to any of the sequences mentioned herein.

The skilled artisan will understand how to calculate the percent identity between two amino acid/polynucleotide/polypeptide sequences. To calculate the percent identity between two amino acid/polynucleotide/polypeptide sequences, the two sequences must first be aligned and then the sequence identity value calculated. The percent identity of two sequences may take different values depending on: (i) Methods for aligning sequences, such as ClustalW, BLAST, FASTA, smith-Waterman (implemented in different programs) or structural alignment from 3D comparison; (ii) Parameters used in the alignment method, such as local to global alignment, pairing score matrices used (e.g., BLOSUM62, PAM250, gonnet, etc.), and gap penalties, such as functional form and constants.

After alignment, there are many different methods to calculate the percent identity between two sequences. For example, the number of identities may be divided by: (i) the length of the shortest sequence; (ii) alignment length; (iii) the average length of the sequence; (iv) the number of gapless positions; or (v) the number of equivalent positions other than overhang. Furthermore, it should be appreciated that the percent identity is also closely related to length. Thus, the shorter a pair of sequences, the higher the sequence identity that happens.

Thus, it should be appreciated that precise alignment of protein or DNA sequences is a complex process. The popular multiplex alignment program ClustalW (Thompson et al 1994,Nucleic Acids Research,22,4673-4680;Thompson et al, 1997,Nucleic Acids Research,24,4876-4882) is the method of choice for generating multiple alignments of proteins or DNA according to the present invention. Suitable parameters for ClustalW are as follows: for DNA alignment: gap open penalty = 15.0, gap extension penalty = 6.66, matrix = identity. For protein alignment: gap open penalty = 10.0, gap extension penalty = 0.2, matrix = Gonnet. For DNA and protein alignment: end= -1, gapdst=4. One skilled in the art will appreciate that it may be necessary to alter these and other parameters to achieve optimal sequence alignment.

Preferably, the percent identity between two amino acid/polynucleotide/polypeptide sequences can be calculated by alignment, i.e., (N/T) 100, where N is the number of positions of the sequence sharing the same residue and T is the total number of positions aligned, including gaps and with or without overhang. Preferably, the overhang is included in the calculation. Thus, the most preferred method for calculating percent identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, such as the parameters described above; (ii) substituting the values of N and T into the following formula: sequence identity= (N/T) 100.

Other methods for identifying similar sequences are known to those of skill in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence that hybridizes under stringent conditions to a DNA sequence or its complement. By stringent conditions, the inventors mean that the nucleotide hybridizes to the filter-bound DNA or RNA in 3 Xsodium chloride/sodium citrate (SSC) at about 45℃and then washed at least once in 0.2XSSC/0.1% SDS at about 20-65 ℃. In addition, substantially similar polypeptides may differ from the amino acid sequences shown, for example, in SEQ ID No. 1 to SEQ ID No. 36 by at least 1, but less than 5, 10, 20, 50 or 100 amino acids.

Due to the degeneracy of the genetic code, it is apparent that any of the nucleic acid sequences described herein may be altered or modified to provide functional variants thereof without materially affecting the protein sequence encoded thereby. Suitable nucleotide variants are those having a sequence that is altered by substitution of different codons within the sequence encoding the same amino acid, thereby producing silent (synonymous) changes. Other suitable variants are variants having homologous nucleotide sequences but comprising all or part of the sequence, which are altered by substitution of different codons encoding amino acids having side chains with similar biophysical properties as the amino acids they replace, to produce conservative changes. For example, small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will thus be appreciated that it will be apparent which amino acids may be substituted with amino acids having similar biophysical properties, and the skilled artisan will also be aware of the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any combination of the aspects described above, except combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to illustrate how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which:

FIG. 1 shows the generation of third generation CD 4-targeted T cells according to an embodiment of the invention. A (1): the figure shows the functional elements comprised in one embodiment of the CAR construct according to the invention (called "CART 4"). The scFv derived from monoclonal antibody Hu5A8 was fused to the CD8 transmembrane domain (TM), CD28 intracellular domain, 4-1BB intracellular domain and CD3 zeta chain. the gene sequences of tgfr (truncated epidermal growth factor receptor) and iC9 (inducible caspase 9) are tagged behind the CAR by self-cleaving 2A linkers. A (2): the figure shows the functional elements comprised in another embodiment of the CAR construct according to the invention (called "cartvb7.1"). The scFv derived from monoclonal antibody 3G5 was fused to the CD8 transmembrane domain (TM), CD28 intracellular domain, 4-1BB intracellular domain and CD3 zeta chain. the gene sequences of tgfr (truncated epidermal growth factor receptor) and iC9 (inducible caspase 9) are tagged behind the CAR by self-cleaving 2A linkers. B: transduced T cells were stained with anti-mouse IgG F (ab)' 2 antibodies and anti-EGFR antibodies. Cell on CD3 ⁺ Proliferation on single lymphocytes, digital representation CAR ⁺ /tEGFR ⁺ Percentage of cells. C: after retroviral transduction of CARs, primary T cells were sampled daily and surface marker stained, including CD3 and tgfr. Blue histograms are the result of non-transduced cells. The percentage of cells positive for CAR and marker is shown. D: survival is defined as exposure to prescribed doses under untreated conditions and under Chemically Induced Dimer (CID) treatment conditionsRatio of percentage of EGFR positivity (a) or EGFR high expression (B) after 24 hours. E: the mean fluorescence intensity of EGFR expression in surviving cells is shown. The data reflects typical results for three replicate samples from different donors. Each sample was repeated three times. Data are expressed as mean ± SEM. The difference between groups was found to be significant by the two-tailed unpaired t test. * P =<0.01；*＝p<0.05。

Figure 2 shows in vitro functional verification of an embodiment of CART 4T cells according to the invention. A: PBMC are activated by Dynabeads human T activator and IL7/IL 15. Activated PBMCs contained two T cell subsets: cd4+ and cd8+ (left). Cells were transduced with CART20 (middle) or CART4 (right) retroviral particles. From the third day after transduction, cells were stained with anti-CD 4 and anti-CD 8 antibodies and analyzed by flow cytometry. B: statistical data for cd4+ ratios are summarized in the figure. The data reflects typical results for five healthy individuals. C: primary cd4+ T cells (left) or cd20+ B cells (right) were co-cultured with autologous CART4 cells, CART20 cells or non-transduced cd8+ T cells for 4 hours. The absolute number of surviving target cells was counted by flow cytometry analysis using Countbright beads. D: two T cell lines, CEM-ss cells, jurkat cells and one B cell line were stained with anti-CD 4 antibodies. CD4 expression levels were assessed by flow cytometry analysis. E: representative results of CART4 cells killing T tumor cells. Each sample was repeated three times. The data reflect the typical results of three independent experiments. F: intracellular cytokine expression of CART4 cells co-cultured with different target cells. Each sample was repeated three times. The data reflect the typical results of three independent experiments.

Figure 3 shows CART4 cell specific killing of cd4+ T tumor cells. PBMCs of ATLL patients were recovered from liquid nitrogen and allowed to stand overnight in an incubator prior to flow cytometry analysis and co-culture experiments. A: PBMCs were stained with anti-CD 4, CD8 and specific TCR Vbeta. Flow cytometry analysis was performed after washing twice with PBS. Resuscitated ATLL (B) or CTCL (C) PBMC were co-cultured with allogeneic CART4 or CART20 cells for 4 hours prior to flow cytometry analysis. Each condition was repeated three times. Data are expressed as mean ± SEM.

Figure 4 shows that CART4 cells effectively mediate anti-leukemia effects in vivo. A: NRG immunodeficient mice were injected 1x10 ⁵ Gluc/GFP transduced CEM-ss cells were then reinfused 4X10 by the retroorbital route ⁶ And (3) T cells. Ntdn=5, cart4n=7. B: each mouse was given 50 μl of peripheral blood and plasma was extracted for determination of luciferase activity. Continuous measurement of luciferase activity showed that CART4T cells inhibited cd4+ leukemia, but NTD cd8+ T cells had no inhibitory effect. C: the overall survival of mice treated with either the indicated CART4 cells or control NTDT cells was analyzed by Kaplan-Meier survival. D: at the endpoint, the mice were dissected. Spleen and bone marrow were ground and stained with anti-CD 4 mAb and DAPI to detect residual tumor cells. Tumor cells were identified as DAPI-CD4+GFP+. E: CD4 expression levels of residual tumor cells in spleen. Gray line-cultured CEMss cells, black line-CEMss cells from NTD control mice, red line-CEMss cells from CART4 treated mice. F: spleen cells were co-cultured with CART4 cells or NTD T cells at a ratio of 1:5 for 4 hours, and then flow cytometry analysis was performed. Data are expressed as mean ± SEM. Significance analysis was performed using a two-tailed unpaired t-test. * =p <0.05。

Figure 5 shows the development of a GMP compliant CAR-T cell manufacturing process. A: time course of CAR-T cell production. Human PBMC were activated with CD3/CD28 Dynabeads and IL7/IL15 in flasks prior to retroviral transduction of CARs. . Two days after transduction, transduced cells were transferred to G-Rex plates (1X 10 ⁶ /square meter). Cytokines were replenished every two to three days until day 12. B: cell expansion during manufacturing. Representative flow charts of CAR transduction rate (C) and differentiation status (D) at day 12. E: statistics of T cell differentiation. CM: a central memory; EM: effect memory. Each sample was repeated three times. Each sample was repeated three times. Data are expressed as mean ± SEM. The data reflects typical results for four healthy individuals.

Fig. 6 shows the generation and functional verification of cartvb7.1. A: transduced T cells were stained with anti-EGFR antibody to detect CAR expression. Cells proliferate on cd3+ single lymphocytes, and numbers represent the percentage of tgfr+ cells. B: endogenous tcrvb7.1+ population detection five days after CAR transduction. C: tcrvb7.1+ primary ATL samples were CFSE stained and mixed with different numbers of effector CAR-T cells. After 6 hours of incubation, cells were collected and stained with DAPI, 3G5 and CD3 antibodies for 15 minutes. A fixed volume of 5uL Countbright beads was added to each sample. The samples were loaded into a flow cytometer for absolute quantification. D: representative results of cartvb7.1 cell killing T tumor cells. Each sample was repeated three times.

FIG. 7 shows the generation process of MAIT-CART cells. A: representative flow cytometry examples of MAIT cell staining. PBMC were stained with BV421 MR1-5-OP-RU tetramer and PE anti-TCR V.alpha.7.2 antibody for 20 min. Cells were washed twice with PBS prior to characterization by flow cytometry. B: gating strategy for flow cytometry sorting of MAIT cells. TCR vα7.2+ cells were isolated from PBMCs by magnetic separation method, followed by staining with BV421 MR1-5-OP-RU tetramer and PE anti-CD 3 antibody for 20 min. Cells were washed twice with PBS and loaded into a moldy cell sorter. Sorting and culturing the MR1-5-OP-RU tetramer positive population. C: amplification curves of MAIT cells cultured in vitro and CD8+ T cells as control. D: after 12-14 days of culture, more than 90% of the expanded MAIT cells have MR1-5-OP-RU tetramer specificity. E: CAR transduced cd8+ T cells and MAIT cells were stained with anti-EGFR flow antibodies to test transduction efficiency.

FIG. 8 shows expanded MAIT and CD 8T cells co-cultured with CFSE-stained CD4+ cell line CEMss at ratios of E: T0:1, 1:1, 3:1 and 5:1. The co-cultivation system was harvested after 20 hours of cultivation. The absolute number of surviving tumor cells was calculated by flow cytometry analysis using Countbright beads. (A) flow cytometry data under 3:1E:T conditions. (B) cytotoxicity statistics. Data are expressed as mean ± SEM.

FIG. 9 shows a first embodiment of the expression vector "CART4" for transduction of MAIT cells.

FIG. 10 shows a second embodiment of the expression vector "CARTVb7.1" for transducing MAIT cells.

FIG. 11 shows the detection of human MAIT cells from Peripheral Blood Mononuclear Cells (PBMC). Lymphocytes were gated and MAIT cells were identified by expression of CD3 and reactivity with the 5-OP-RU/MR1 tetramer (A) or expression of CD161 and TCRVα7.2 (B).

FIG. 12 shows the isolation of human MAIT cells from Peripheral Blood Mononuclear Cells (PBMC). After isolation of vα7.2 expression by magnetic beads, the vα7.2 positive cell population was enriched from 2.2% (a) to >97%. MAIT cells were sorted according to reactivity with 5-OP-RU/MR1 tetramer (B).

FIG. 13 shows the production of CAR-MAIT cells. After 12-14 days of culture, more than 90% of the expanded MAIT cells have MR1-5-OP-RU tetramer specificity (A). CAR transduced cd8+ T cells and MAIT cells were stained with anti-EGFR flow antibody to detect transduction efficiency (B).

Figure 14 shows that CAR-MAIT4 cells exhibit potent anti-leukemia function in vivo. A: NSG immunodeficient mice were given 1X10 intravenous injections ⁶ Gluc/GFP transduced CEM-ss cells, then injected intravenously 4X10 ⁶ Individual CARs transduce cells. Bioluminescence imaging was performed twice weekly until day 45 after tumor injection. B: overall survival of the indicated CAR-transduced cell treated mice was obtained by Kaplan-Meier survival analysis. C: bioluminescence imaging (BLI) of mice 40 days after tumor injection. D: irradiation of individual mice at day 40. n=5 or 6 mice/group. * By t-test P<0.05.Ph, photons; sr, sphericity.

FIG. 15 shows enrichment of MAIT cells in PBMC. PBMC were stimulated with MR1/5-OP-RU composite beads (bead to cell ratio 1:1) or 5-OP-RU antigen (10 nM) in the presence of the different cytokines shown in the tables for 6 days. The fold increase in MAIT cells was calculated by dividing the frequency of viable MAIT (CD3+Va7.2+CD161+) cells on day 6 by the original frequency of MAIT cells on day 0. The first 5 groups are marked with orange colors (i.e., conditions 1, 3, 11, 12, and 13). The combination of IL-12, IL-18 and IL-23 maximizes the fold increase in MAIT cells in PBMC.

Examples

T cell therapies based on Chimeric Antigen Receptors (CARs) have met with great success in the treatment of B cell malignancies by targeting pan B cell specific antigens. However, similar strategies for T cell lymphomas have not been achieved to date, mainly due to the potentially serious toxicity associated with overall T cell depletion and low normal T cell dysfunction/frequency in T lymphomas compared to B cell malignancies. To overcome these limitations, the inventors designed two novel CAR constructs, the first referred to herein as "CART4" with specificity for the pan-T cell marker (CD 4) and the second referred to as "cartvb7.1" with specificity for the TCR-Vb isotype chain. Both CAR constructs contained one or two safety switches selected from truncated epidermal growth factor receptor (tgfr) and inducible caspase 9 (iC 9). However, as shown in fig. 1, both safety switches are shown. The inventors investigated whether mucosal-associated constant T (MAIT) cells with low alloreactivity would exhibit similar anti-tumor killing activity to conventional T cells after transduction with CAR constructs.

In addition, it is well known that MAIT cells are a subset of congenital T cells, defined as CD3+TCRVa7.2+CD161+ cells, recognizing the MHC class I molecule MR1. Previous studies have shown that MAIT cells can be expanded in vitro, but require the presence of allogeneic feeder cells. However, one problem with this approach is that it is difficult to mass produce and quality control. In this study, the inventors have now developed an efficient method for in vitro expansion of MAIT cells by first stimulating PBMC with antigen (5-OP-RU) loaded MR1 tetramer beads or with 5-OP-RU alone, in vitro culture in the presence of a combination of cytokines (IL-2, IL-7, IL-15, IL-12, IL-18 and IL-23) for up to 6 days. MAIT cells were then isolated by MACS or FACS sorting, followed by further expansion by anti-CD 3/CD28 beads for CAR-based treatment as described in the previous examples.

Materials and methods

Construction of CAR plasmids

DNA fragments encoding scFv of Hu5A8 and Leu16 and human igκ leader were synthesized by Genewiz. NcoI and NotI were used to cleave these fragments and MSCV CAR-expressing retroviral vectors. The MSCV CAR expression vector is modified by MSCV-IRES-GFP vector (Addgene), and IRES-GFP region is replaced by human CD8 transmembrane domain and third generation CAR intracellular signaling domain (co-stimulatory domain of CD28 and 4-1BB, CD3 zeta signaling domain). the sequence of tgfr was obtained from US 8802347B2, deleting domains I and II of the extracellular portion and intracellular domain of the human EGFR protein. tEGFR was synthesized by Genewiz and has a self-cleaving T2A sequence and a leader peptide for the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. The DNA sequence of iC9 is provided by Lishan Su professor (Church mountain division of university of North Carolina) friends. The DNA fragment of iC9 consists of truncated caspase 9, including the large and small subunits of the caspase molecule, linked to a 12-kDa human FK506 binding protein (FKBP 12) via a short Gly-Gly-Gly-Ser (GGGS) flexible linker.

Production of retroviral vectors

Plat-GP cells (Cellbiolabs) were transfected with MSCV-retroviral plasmid and pCMV-VSVG vector (adedge) by 7ul X-tremgene HP transfection reagent (Roche) to generate viruses with VSV envelope. To generate high titers of CAR-encoding retrovirus supernatant, a stable virus-producing cell line was subsequently performed with PG-13 (ATCC). PG-13 cells were transduced with Plat-GP virus supernatant containing 8ug/ml polybrene (Sigma). The plates were wrapped with preservative film and centrifuged at 1000g for 2 hours at 32 ℃. To generate high titers of viral particles, confluent cells were passaged by trypsinization. After 24 hours, the supernatant was collected. After centrifugation at 300g for 5 minutes, the medium was dispensed and stored at-80 ℃.

Primary T cells and tumor cells

Peripheral Blood Mononuclear Cells (PBMC) of healthy donors were isolated by Ficoll-Paque PLUS density gradient centrifugation (GE Healthcare) for CAR-T cell engineering. T lymphoma cell lines derived from ATL or CTCL patients were cultured in D10 medium (DMEM with 10% fetal calf serum, 100IU/mL penicillin, 100. Mu.g/mL streptomycin and 2mM L-glutamine), and T leukemia cell lines (Jurket or CEM) were cultured in R10 medium (RPMI 1640 with 10% fetal calf serum, 100IU/mL penicillin, 100. Mu.g/mL streptomycin and 2mM L-glutamine).

Isolation and expansion of MAIT cells

MAIT cells were isolated from healthy PBMC by a two-step procedure using anti-human TCR V.alpha.7.2 antibody (Biolegend, cat # 351724), microbeads (Miltenyi, cat # 130-090-485), and then BV421 MR1-5-OP-RU tetramer (supplied by Jim McCluskey professor of the university of Meerce, australia). Briefly, TCR vα7.2+ t cells were isolated from PBMCs according to the manufacturing procedure using biotinylated anti-human TCR vα7.2 antibody and an avidin microblads kit (Miltenyi). The MAIT cells were then isolated from TCR V.alpha.7.2+ T cells by staining BV421 MR1-5-OP-RU tetramer and FACSMELID cell sorter (BD) for FACS sorting.

MAIT cells were isolated from PBMC by a two-step procedure. PBMC cells were counted and diluted to 1X 10 in PBS/EDTA buffer in 15mL tubes ⁸ cells/mL. Every 10 ⁸ mu.L of biotin anti-human TCR V.alpha.7.2 antibody (Biolegend, cat # 351724) was added to each cell and incubated at 4℃for 20 minutes. Cells were washed by adding 10 volumes of PBS/EDTA buffer and centrifuged at 300 Xg for 10 min. The supernatant was completely aspirated. Every 10 ⁸ mu.L of PBS/EDTA buffer and 200. Mu.L of avidin MicroBeads (Miltenyi, cat# 130-090-485) were added to each cell. Mix well and incubate at 4℃for 15 min. Cells were washed by adding 10 volumes of PBS/EDTA buffer and centrifuged at 300 Xg for 10 min. The supernatant was completely aspirated. Up to 10 cells were resuspended in 1mL PBS/EDTA buffer. The MS column (Miltenyi) was placed in the magnetic field of a suitable MACS separator. The column was washed with 500. Mu.L of PBS/EDTA buffer. The cell suspension was applied to the column. The column was washed with 3X 500. Mu.L PBS/EDTA buffer. 1mL of PBS/EDTA buffer was added and the cells remaining outside the magnetic field were eluted. TCR V.alpha.7.2+ cells were collected and stained with APC anti-human CD3 (Biolegend) and BV421 MR1-5-OP-RU tetramer (1:500) at 4℃for 30 min. Cells were washed by adding 10 volumes of PBS/EDTA buffer and centrifuged at 300 Xg for 10 min. The supernatant was completely aspirated. Every 10 ⁸ 1mL of PBS/EDTA buffer was added to each cell. CD3+MR1-5-OP-RU+ cell populations were flow sorted by FACSMEbody cell sorter (BD).

Activation and expansion of MAIT cells

The sorted MAIT cells were counted and resuspended to 10 in R10 medium (90% RPMI+10% FBS+1% penicillin/streptomycin+2 mM L-glutamine) ⁶ Individual/mL. Cells were activated using Dynabeads human T activator CD3/CD28 (Life Technologies company) and 100IU/mL IL-2 was used in 24 well plates in an incubator at 37℃such that the bead to cell ratio reached 1:1. Two days after transduction, cells were harvested and transferred to 6-well plates. Refreshing R10 to 0.5-1x 10 ⁶ And each mL. The cells were refreshed every 2-3 days with R10 medium.

Production of CAR-T or CAR-MAIT cells

Purified PBMC or MAIT cells were stimulated with Dynabeads human T activator CD3/CD28 (Life Technologies Co.) in R10 medium containing 100IU/mL IL-2 for 48 hours. Activated cells were then transfected with retrovirus encoding the CAR construct and cultured in R10 for an additional 48 hours. Prior to harvest, CAR-T or CAR-MAIT cells were maintained in G-Rex six well plates (Wilsonwolf) in the presence of recombinant IL7 and IL15 (Miltenyi) for an additional 7 days.

Production of CAR-T or CAR-MAIT cells

Retroviral transduction was performed 48 hours after T cell activation. The transduction step may be repeated after 24 hours if necessary to increase transduction efficiency. Will be 10x10 ⁶ The individual cells were transferred to G-Rex six well plates (Wilsonwolf) and further cultured with 110mL of R10 medium. Cytokine IL7/15 was supplemented every two or three days. Cells were cultured in G-Rex for one week prior to harvest.

Co-culture cytotoxicity assay

The non-radioactive killing assay was performed as reported previously (Rowan et al, 2014). Briefly, target cells were stained with 1uM CFSE (Biolegend) for 15 min at 37 ℃. After 3 washes in PBS 100000 target cells were mixed with CAR-T or CAR-MAIT cells in a ratio of 1:1, 1:3, 1:5. 200ul of the co-cultures were incubated in an incubator at 37℃for 4 hours. To the samples were added 1ul DAPI and 5ul Countbright beads (BD Biosciences). Samples were collected by flow cytometry at a constant rate. The number of surviving target cells was calculated as: number of cells in tube = (number of cells collected/number of beads collected) x total number of beads added to tube.

Intracellular cytokine staining

CART4 or CART 20T cells and specific target cellsCo-culture was performed in 96 well U-bottom plates. All cells were at 2x10 ⁵ Each well was inoculated in 200 uL/well of R10 medium. T cells cultured alone served as negative control and T cells cultured were stimulated in combination with 10ug/ml PMA and 10ug/ml ionomycin (Biolegend) served as positive control. After 1 hour of incubation, 10ug/mL briafectin A (Biolegend) was added to all wells. The co-cultivation system was harvested after five more hours of cultivation. Cells were surface marker stained with antibody for 30 minutes in the dark. Cells were fixed with 4% paraformaldehyde solution (Biolegend) for 15 min at room temperature and then rinsed with PBS. After re-washing with the fixation buffer, the cells were resuspended with a mixture containing the intracellular staining antibodies. The cells were incubated at 4℃for 30 min and then washed with fixation buffer. They were analyzed by flow cytometry and Fluorescence Minus One (FMO) control to determine the expression levels of IFN- γ and TNF- α.

In vitro suicide test

Transduction of CART4 or CART4 w/o iC9 constructs with retroviruses generates CART4 with or without iC9 cells. CAR-T cells remained expanded for 5 to 7 days after transduction. Caspase-inducing drug (CID), B/B homodimer AP20187 (Clontech Laboratories) was added to T cell cultures at different concentrations. After 24 hours, CID-induced apoptosis was assessed using annexin-v/7-AAD (BD Biosciences) staining and flow cytometry analysis. Surviving cells were quantified by counting beads (BD Biosciences). The survival index calculation method is as follows: number of surviving tEGFR+ cells/number of surviving tEGFR+ cells in untreated control samples.

In vivo mouse xenograft experiments

In vivo experiments of T leukemia cell lines were performed using NRG mice (Jackson Laboratory) of 6 to 8 weeks of age. Will co-express Gaussia luciferase and EGFP 0.5x10 ⁶ CEM-ss cells were injected into mice by reverse-track. The mice were then randomized prior to T cell injection. PBMCs from healthy donors are activated and transduced to generate CART4T cells or non-transduced T cells. On the day of transfusion, non-contacted microbead human CD 8T cell kit (Miltenyi) was used to extract from CART4T cells or non-transduced T cells according to the manufacturer's instructionsCART4 cd8+ T cells and non-transduced cd8+ T cells were sexually isolated. Will be 4×10 ⁶ The isolated cells were washed twice with PBS, resuspended in 100ul and infused into xenograft mice by retroorbital injection. 30-50ul of peripheral blood was withdrawn weekly through the empty tail. After centrifugation at 500g for 5 minutes, the plasma was separated and luciferase activity was measured according to the manufacturer's instrument (Thermo Fisher Scientific). After erythrocyte lysis, cells were resuspended with 100ul of antibody premix for surface marker staining, including human CD45, mouse CD45, human CD3, human CD8, human EGFR, and live/dead dyes. The cell subpopulation composition was analyzed using a flow cytometer (BD AriaIII). Mice were closely monitored in all studies described herein. Mice were euthanized when they exhibited one of the following symptoms: initial weight loss was more than 20%, a pronounced sleepiness, a humpback posture, severe diarrhea or severe dermatitis.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software version 6.0 (GraphPad software). Two sets of data were compared using a two-tailed unpaired t-test. * P <0.05, < P <0.01, < P <0.001. All data with error bars are expressed as mean ± Standard Error of Mean (SEM). P values less than 0.05 are significant. Data were analyzed using GraphPad Prism software (version 8).

1.Specific method

1.1.Detection of MAIT cells

1.1.1. Preparation of human Peripheral Blood Mononuclear Cells (PBMC)

1.1.1.1. Peripheral blood from 5mL volunteer donors was transferred to heparinized tubes.

1.1.1.2. Whole blood was diluted with an equal volume of PBS.

1.1.1.3. 5mL of Histopaque-1077 was placed in a 15mL centrifuge tube. Carefully add 10ml of diluted blood solution gently to Histopaque-1077 along the tube wall; without disrupting the liquid interface.

1.1.1.4. Centrifuge at 400x g for 30 minutes at room temperature.

Note that: ensuring that the centrifuge braking and acceleration settings are at a minimum setting, severe braking and acceleration may affect layer separation.

1.1.1.5. After centrifugation, the upper layer was carefully aspirated with a Pasteur pipette to within 0.5cm of the opaque interface containing monocytes. The upper layer is discarded.

1.1.1.6. Harvested PBMCs were washed twice with 10ml PBS by centrifugation at 250x g for 10 minutes.

1.1.1.7. PBMC pellet was resuspended in RPMI1640 medium.

Preparation of 5-OP-RU/MR 1-tetramer labeled with BV421

1.1.2.1. 6.8 μl of 0.5mg/ml streptavidin-BV 421 was diluted into 10.2 μl PBS and mixed well.

1.1.2.2. 1/10 of the streptavidin-BV 421 solution (1.7. Mu.l) was added to 18. Mu.l of MR1-5-OP-RU solution (5. Mu.g) every 10 minutes and mixed with a pipette and incubated in the dark at room temperature between the two steps.

1.1.2.3. The BV 421-labeled 5-OP-RU/MR1 solution was maintained at 4 ℃.

The tetramer should be titrated for use; a dilution of 1:500 is usually sufficient.

1.1.3. Detection of human MAIT cells by flow cytometry

Human MAIT cells can be detected by flow cytometry, by 5-OP-RU loaded MR1 tetramer or co-stained with CD161 and TCR V.alpha.7.2 chain antibodies. In general, MAIT cells account for 0.1-10% of CD3+ T cells in peripheral blood. 1.1.3.1. 1X 10 staining buffer per 100. Mu.l FACS ⁶ The concentration of individual cells resuspended PBMCs.

1.1.3.2. FACS antibodies and/or 5-OP-RU/MR1 tetramers are added to the samples.

For detection of human MAIT cells, two methods can be used:

a) Tetramer staining: human 5-OP-RU MR1 tetramer (1:500) and APC-H7 conjugated anti-human CD3 (1:200) were labeled with BV 421.

b) Substitution markers: PE conjugated anti-human TCR Va7.2 (1:200), APC-H7 conjugated anti-human CD3 (1:200) and FITC conjugated anti-human CD161 (1:200).

1.1.3.3.4 ℃for 30 minutes in the absence of light.

1.1.3.4. Cells were washed with FACS staining buffer by centrifugation at 300x g for 5 min at 4 ℃ and resuspended with 300 μl FACS staining buffer.

1.1.3.5. MAIT cells were analyzed by flow cytometry (see FIG. 11).

1.2.Isolation of MAIT cells

Magnetic bead separation of vα7.2+ cells.

1.2.1.1. PBMCs were collected and cells were washed with binding buffer. The supernatant was discarded and concentrated at 1X 10 ⁷ 100 μl of MACS buffer resuspended cell pellet. PE anti-human TCR V.alpha.7.2 antibody (1:100) was added. Mix well and incubate on ice for 30 minutes.

1.2.1.2. The cells were washed once with MACS buffer by centrifugation at 300x g for 5 min.

1.2.1.3. MACS buffer at 10 ⁷ Cells were resuspended at a concentration of/80. Mu.l. Every 10 ⁷ Mu.l of anti-PE microbeads were added to each cell, and incubated on ice for 20 min.

1.2.1.4. Cells were washed once with 10 volumes of MACS buffer. Centrifuge at 300x g for 5 minutes. Resuspended in 1ml MACS buffer.

1.2.1.5. The MS column was pre-washed with 1ml MACS buffer and assembled on a magnet. The cells were applied to the column and the column was washed 3 times with 1ml MACS buffer each time.

1.2.1.6. The column was removed from the magnet and bound cells eluted in 1ml MACS buffer.

Flow sorting of MAIT cells

1.2.2.1. Magnetically isolated cells were collected and centrifuged at 300x g for 5 minutes.

1.2.2.2. MACS buffer at 10 ⁷ Cells were resuspended at a concentration of 100. Mu.l. BV 421-labeled human 5-OP-RU MR1 tetramer (1:500) and APC-H7-conjugated anti-human CD3 (1:200) were added. Incubate on ice for 20 minutes.

1.2.2.3. Cells were washed once with 10 volumes of MACS buffer. Centrifuge at 300x g for 5 minutes. Resuspended in 2ml MACS buffer.

1.2.2.4. The BD Prodigy sorter was opened and the cell samples loaded. Cd3+ tetramer+ cell populations were sorted (see fig. 12).

1.3.Activation of MAIT cells

1.3.1. Sorted MAIT cells were collected and centrifuged at 300x g for 5 min.

1.3.2. The supernatant was discarded and resuspended to 10 in R10 medium ⁶ Individual cells/ml.

1.3.3. Dynabeads human T activator CD3/CD28 was resuspended by vortexing for 30 seconds.

1.3.4. The required volume of Dynabeads was transferred to a tube.

1.3.5. An equal volume of buffer was added and vortexed for 5 seconds. The tube was placed on a magnet for 1 min and the supernatant was discarded.

1.3.6. The tube was removed from the magnet and the washed Dynabeads were resuspended in R10 medium.

1.3.7. The required volume of Dynabeads was added to the cell suspension and 100IU/ml IL-2 was added to a 24-well plate in an incubator at 37℃so that the bead to cell ratio reached 1:1.

1.4.Retroviral transduction of MAIT cells

1.4.1. Retroviral transduction was performed 48 hours after MAIT cell activation.

1.4.2. One day prior to transduction, retroNectin coated plates were prepared. 15 μg of retroNectin was added to 1ml PBS.

Mix well and add to one well of a 24-well plate without tissue culture treatment.

1.4.3. The plates were wrapped with preservative film and stored overnight in a refrigerator at 4 ℃.

1.4.4. On the day of gene transfer, unbound retroNectin was removed from the wells. Wash twice with 2ml PBS. Avoiding sufficient drying.

1.4.5. The retrovirus supernatant was thawed in a water bath at 37 ℃. 1ml of virus supernatant was transferred to each well of the RetroNectin coated plate.

1.4.6. The plates were wrapped with preservative film and centrifuged at 1000x g for 2 hours at 32 ℃.

1.4.7. During centrifugation, activated MAIT cells are collected. The cells were resuspended to a concentration of 1X 10 with fresh R10 medium containing 100IU/ml IL-2 ⁶ /ml。

1.4.8. After the end of the rotation, the supernatant on the plate was discarded. 1ml of cell suspension was added to each well.

1.4.9. Plates were centrifuged at 500x g for 10 minutes.

1.4.10. The plates were returned to the 37℃incubator.

1.4.11. The transduction step is repeated as necessary to increase transduction efficiency.

Note that: transduction efficiencies can be measured by flow cytometry 48 hours after transduction.

1.5.Expansion of CAR-MAIT cells

1.5.1. Two days after retroviral transduction, cells were harvested and counted by a hemocytometer.

1.5.2. Will be 1x 10 ⁷ Individual cells were transferred to one well of a Grex6M well plate. 130ml of fresh R10 medium containing 100IU/ml IL-2 was added and the plates were returned to the incubator.

1.5.3. IL-2 was refreshed every three days to a final concentration of 100IU/ml.

1.5.4. After 8-12 days of culture, CAR-MAIT cells can be harvested (see FIG. 13).

Note that: the amplified CAR-MAIT cells can be used for phenotypic testing, functional assays, or liquid nitrogen freezing.

Results

Example 1: production of CD 4-targeting T cells and TCR Vbeta 7.1-targeting T cells

Referring to fig. 1A (1) and (2), functional elements included in two different embodiments illustrating CAR constructs according to the invention are shown.

In each embodiment, the construct is flanked by upstream and downstream Long Terminal Repeats (LTRs). The 5 'promoter is located downstream of the 5' LTR, and may be a PGK promoter. The promoter has an SP at its 3' end, an Ig kappa signal peptide, which directs the fusion protein to the T cell outer membrane. An scFv region is provided 3' to the SP, which includes an upstream VL (variable light chain) sequence, a central G4S sequence, and a downstream VH (variable heavy chain) sequence. In one embodiment (as shown in fig. 1A (1)), the VL and VH sequences may be Hu5A8 light chain variable region and heavy chain variable region to bind CD4 antigen. In other embodiments (as shown in fig. 1A (2)), the VL and VH sequences may be 3G5 light chain variable regions and heavy chain variable regions to bind TCR-vb7.1.

The scFv region has a CD8a hinge and a Transmembrane (TM) domain 3' to the scFv region for CAR display and anchoring. The hinge and the 3' end of the TM are provided with intracellular domains, including the signal domains of the CD28, 4-1BB and CD3 zeta chains, which trigger intracellular signaling pathways. The P2A self-cleaving peptide is located 3 'of the zeta chain and 5' of the truncated EGFR (EGFRt) for tracking and acting as a first safety switch. The second P2A self-cleaving peptide is located at 3 'of EGFRt and 5' of inducible caspase 9 (iC 9), acting as a second safety switch. The construct includes woodchuck hepatitis regulatory element (WPRE) (see plasmid of FIGS. 9 and 10) that enhances expression, and finally the terminal 3' LTR.

The DNA sequence of the humanized 5A8 (Hu 5 A8) mouse IgG antibody with immunospecific for human CD4 was found in CN 103282385. Hu5A8, also known as TNX-355 or Ai Bali bead mab (ibalizumab), is widely evaluated in phase I and phase II clinical trials to inhibit HIV entry by blocking the HIV binding site of the CD4 molecule. As a control, VH and VL chains of an anti-CD 20 monoclonal antibody (Leu 16) were also synthesized and their efficacy was assessed in preclinical and clinical CAR-T studies. The scFv fragment was cloned into the backbone of the third generation CAR plasmid, with a CD8 transmembrane domain, a CD28 intracellular domain, a 4-1BB intracellular domain, and a CD3 zeta chain. The use of third generation CARs has been demonstrated to be superior to first and second generation CARs. As an option, the utility of tgfr as an in vivo tracking marker and as the first safety switch to ablate engineered CAR-T cells was examined. Thus, the residual tgfr sequence is linked to the CAR sequence by a T2A-ribosome jump sequence.

CAR-T cells can sometimes remain in patients for decades, as is the case with anti-CD 19 and anti-HIVCAR assays. Unlike B cell dysgenesis, long-term cd4+ T cell dysgenesis can be life threatening. Thus, it is necessary to establish a safe method for removing the CART4 cells of the invention from the patient in case of emergency after tumor or virus elimination or due to serious side effects during CAR-T treatment. Dimerization drug-induced caspase 9 (iC 9) suicide switch based on fusion of human caspase 9 with mutated human FK506 binding protein (FKBP), conditional dimerization can be achieved in the presence of the small molecule chemical drug AP20187 (termed caspase-induced drug, CID). The use of iC9 has been demonstrated to be safe and effective in clinical trials of semi-syngeneic HSC transplantation. Thus, a gene fragment comprising CD4 CAR, tEGFR and iC9 was subsequently synthesized (FIG. 1 A.1) and inserted into a retroviral MSCV (murine Stem cell Virus) vector (as shown in FIG. 9; 10,348 bp). Furthermore, a gene fragment comprising TCR Vmeeting 7.1 CAR, tEGFR and iC9 was subsequently synthesized (FIG. 1 A.2) and inserted into a retroviral MSCV (murine Stem cell Virus) vector (as shown in FIG. 10; 10,347bp).

To confirm co-expression of CAR and tggfr, human PBMCs were activated with Dynabeads human T activator and genetically engineered to express CAR/tggfr/iC 9 gene constructs by retroviral transduction of the plasmids shown in fig. 9 and 10. Indeed, the expression levels of CAR and tgfr are closely related and after surface staining with a mouse scFv specific anti-mouse IgG F (ab') 2 antibody binding EGFR specific antibody, a double positive cell population can be detected (fig. 1B). As T cells expand, the tgfr+ cell population maintains its proportion around 50% of the total cell number (fig. 1C).

To evaluate the efficiency of the in vitro iC9 safety switch, CART4 variants (CART 4 w/o iC 9) without iC9 gene were cloned as controls. T cells transduced with CART4 or CART4 without iC9 construct were exposed to increasing concentrations of CID AP20187 (0.1 nM to 100 nM) for 24 hours. Flow cytometry analysis was performed using 7AAD and annexin-V to understand cell death. The percent of tgfr positivity in the surviving cell population decreased with increasing CID concentration. A single 100nM dose of CID resulted in the elimination of 69.1% of tggfr high cells (fig. 1D). Consistent with observations from other studies, the escape killed cells were those expressing low levels of transgene, and the average fluorescence intensity (MFI) of tgfr was reduced by 50% after CID (fig. 1E). Thus, non-reactive T cells express iC9 insufficient to activate CID function. For clinical use, CAR-T cells may need to be sorted prior to administration to obtain adequate transgene expression.

Example 2: in vitro function of CART 4T cellsVerification

Within four days after CAR transduction, cd4+ T cells were almost completely depleted compared to the non-transduced (NTD) and CART20 controls, with about 45% of cells still being CD4 positive (fig. 2A). These data indicate that CART4 cells have strong anti-CD 4 activity during T cell expansion.

Co-cultures were established against autologous primary healthy donor PBMC. CFSE labeled autologous PBMCs were co-cultured with cd8+ CART4 cells or CART20 cells. In both cases, CART4/20 cells were able to produce high levels of cytotoxicity to the respective target cells. During 4 hours co-culture 94% of CD4+ cells in PBMC were lysed by CART4 cells at an E:T ratio of 3:1. However, CART4 has no specific T cytotoxicity for cd20+ cells compared to NTD T cells (fig. 2C).

To further evaluate the function of CART4 cells, the inventors tested the antitumor efficacy of CART4 cells using Jurkat cell lines and CEM-ss cell lines. Jurkat and CEM-ss cell lines are T cell lines originally established from the peripheral blood of patients with T cell leukemia or human T4 lymphocytic leukemia. Both cell lines expressed CD4, whereas CEM-ss cell lines expressed higher levels of CD4 (fig. 2D). In fact, CART4 cells target T tumor cell lines according to CD4 expression levels. CART4 cells were successfully depleted of CEM-ss cells at a 5:1 E:T (effector: target) ratio after short term culture. As a control, CART4 cells were also tested for their activity on CD 4-lymphoma cells (a human B cell line that does not express CD4, BCL) (fig. 2D). Flow cytometry analysis showed that CART4 cells failed to target BCL (fig. 2E). Furthermore, CART4 cells cultured with CD4+ tumor cells showed significant IFN-gamma and TNF-alpha responses by intracellular cytokine staining (FIG. 2F). Thus, these data demonstrate a strong dose-dependent response of CART4 to CD4 expression. No killing was observed when CART4 cells were cultured with CD4 negative cells. Thus, these results indicate that CART4 cell ablation is specific for CD 4.

Example 3: CART4 cell specific killing CD4+ T tumor cells

To examine the function of CART4 on patient samples, PBMCs from ATLL patients were thawed and phenotyped. CD4 expression rates were 67.4% to 97.7% for all samples. Most cd4+ cells expressed a unique T cell receptor (TCR Vbeta) β chain, indicating clonal development of T cell leukemias 202-204 (fig. 3A). After 4 hours of co-culture of ATLL patient samples with CART4 cells, cd4+ malignancy was rapidly and clearly ablated as quantified by flow cytometry analysis. About 80% ablation was observed for all ATLL co-cultures, consistent with the ablation of the parent T cell line previously shown (fig. 3B). The study also used samples from six CTCL patients. Also, CART4 cells were observed to have strong cytotoxicity against freshly thawed primary CTCL cells, and after co-culture for 4 hours, malignant T cells were reduced by about 60% to 80% (fig. 3C). Thus, CART4 cells can effectively eliminate invasive cd4+ T malignancies isolated directly from patient samples. These results indicate that CD4 is a very promising therapeutic target for cd4+ T malignancies.

Example 4: CART4 cells effectively mediate internal anti-leukemic effects

To evaluate antitumor activity in vivo, the inventors developed a xenogenic mouse model using CEM-ss cell lines expressing Gaussia luciferase. First a single dose (4 x10 ⁶ ) The CART4 cells of (c) tested the ability of CART4 cells to delay the appearance of NRG mouse leukemia. Flow cytometry analysis indicated that approximately 50% of cells prior to injection expressed anti-CD 4 CAR. Mice received retroorbital injection of CEM-ss cells. Four days after tumor implantation, leukemic mice were subjected to single dose retroorbital injection of CART4 cells or NTD cd8+ T cells (fig. 4A). Tumor burden was monitored by weekly measurement of luciferase activity in peripheral blood. The infused CART4 cells were effective in preventing leukemia progression (fig. 4B) and significantly prolonged median survival in mice (38 days for control, 60 days for CART4, p=0.026 by Mantel-Cox log rank test) (fig. 4C). Indeed, by flow cytometry analysis, egfp+ tumor progression was significantly delayed in spleen and bone marrow by endpoint (fig. 4D).

Although recurrent tumor cells retained CD4 expression, the expression level was reduced to about 40% MFI compared to the control group (fig. 4E). However, this down-regulation was insufficient to impair the ability of CART4 cells to eliminate recurrent tumors (fig. 4F). This result suggests that the primary cause of tumor recurrence is the lack of persistence of CAR-T cells, rather than antigen escape.

Example 5: method for developing GMP (good manufacturing practice) standard-compliant CAR-T cell production method

To assess the scalability of CAR-T cells and simplify CAR-T cell manufacturing, an optimized standard procedure was established using a gas-permeable static cell culture system (G-Rex) for CAR-T cell manufacturing (fig. 5A). The G-Rex system contains a layer of silicone film at the bottom of the plate. By gas exchange over the membrane (including O ₂ And CO ₂ ) The depth of the medium can be increased, providing more nutrients and diluting the waste. After activation and transduction of PBMC, 10X10 ⁶ Individual cells were transferred to G-Rex six well plates for further culture. The cells were supplemented with cytokines IL-7 and IL-15 every two to three days. From the initial 2x10 cells ⁶ Amplified to more than 3x 10 ⁸ And, by a factor of 150 within 15 days (fig. 5B). Next, the transduction efficiency of the final product after T cell expansion in the G-Rex system was determined. The final transduction efficiency of CART4 was 57.6% ± 7.1%, similar to cells generated in a conventional flask (53.7% ± 5.3%), as shown in fig. 5C. As expected, the endogenous cd4+ population in the final product was fully depleted, indicating that CAR-T cells have anti-CD 4 activity.

Interestingly, CAR-T cells generated in G-Rex exhibited a differentiation preference for the central memory phenotype. Evaluation of memory markers CD45RO and CD62L showed a higher proportion of CD45RO CD62L double positive cells (77% 7.1% vs 41% 5.5%) compared to cells cultured in conventional flasks (fig. 5D). CD45RO CD62L double positive cells are central memory T cells, thought to be necessary for long-term persistence in the body. Thus, this biological process optimization approach increases the ratio of cell yield and central memory phenotype while reducing the number of technician interventions and the cost of CAR-T manufacture.

Example 6: production of TCR vbeta7.1 specific CAR-T cells

T cell malignancies typically develop from a single type of monoclonal cancer cell that expresses a unique TCR. A variety of antibodies to the variable (V) region of the tcrp (vβ) chain have been provided in a direct conjugated polychromatic format, 22 of 25 vβ families can be assessed, covering 75% of the normal circulating T cell lineage. Thus, the inventors believe that TCR vβ is a potential target for CAR-T therapy against T cell malignancies. To develop TCR vβ targeting CAR-T (cartvb7.1) cells, the inventors cloned the scFv region of hybridoma cell 3G5 onto a CAR construct, which hybridoma cell was able to produce a monoclonal antibody against human TCR vβ 7.1 (Margret calam doctor from Andrew laboratories, oxford university), as shown in fig. 1A (2). Five days after CAR transduction, the endogenous TCR vβ7.1+ cell population was almost completely depleted, with about 1.2% of the cells remaining TCR vβ7.1 positive compared to CART20 control (fig. 6B). These data indicate that CAR-T cells have potent activity against TCR vβ7.1 during T cell expansion.

To further evaluate the function of cartvb7.1 cells, the inventors tested anti-tumor efficacy using tumor cells isolated from ATL patients diagnosed with tcrvβ7.1 positive tumors. In fact, after 6 hours of co-culture of ATL patient samples with cartvb7.1 cells, cd4+ malignancies were rapidly and unequivocally ablated by flow cytometry analysis of the quantified results. About 60% ablation was observed for all ATL co-cultures (fig. 6C, D). These results indicate that TCR vβ is a promising therapeutic target for T malignancy.

Example 7: development of CAR-MAIT cells

Currently, most CAR-T therapies utilize autologous conventional cd3+ T cells. However, immune cells of cancer patients may be hypofunctional or of a small number. In particular, the expansion and genetic engineering of PBMCs of T malignancy patients is risky because tumor cells can be engineered with CARs. Thus, there is a need to develop an immunotherapy that can produce third party allogeneic cells. Here, the inventors developed a two-step method to isolate mucosal-associated constant T cells (MAIT cells) from PBMCs by combining magnetic separation with flow cytometry sorting. The first step was to increase the percentage of MAIT cells from 0.74% to 33.3% after isolation based on TCR V.alpha.7.2 expression (FIG. 7A, B). The next step of flow sorting can further increase the MAIT purity to 95%. The sorted cells were activated with Dynabeads human T activator CD3/CD28 and expanded in the presence of cytokine mixtures (IL-2, IL-7 and IL-15). The amplification method achieved about 100-fold amplification in 12 to 14 days (fig. 7C). At harvest, 90.9% of the expanded cells maintained specificity for the MR1-5-OP-RU tetramer (FIG. 7D). In addition, amplified MAIT cells can be successfully CAR engineered by retroviral transduction (FIG. 7E). CAR-transduced MAIT (CAR-MAIT) cells had comparable cytotoxic capacity to conventional CAR-T cells (fig. 8).

Example 8: CAR-MAIT cells effectively mediate in-vivo anti-leukemia effects

To evaluate the in vivo antitumor function of CAR-MAIT cells, the inventors used a single dose (4 x10 ⁶ ) Is tested for the ability of anti-CD 4 CAR-MAIT (CAR-MAIT 4) cells to delay progression of NRG mouse leukemia. Mice received intravenous CEM-ss cells. Four days after tumor implantation, leukemia mice were given single dose intravenous injection of CAR-MAIT4 cells or CART4 cells (fig. 14A). anti-CD 20 CAR-MAIT (CAR-MAIT-Ctrl) cells or anti-CD 20 CART (CART-Ctrl) cells were used as control groups. Tumor burden was monitored by weekly measurement of luciferase activity. The infused CAR-MAIT4 cells and CART4 cells were effective in preventing leukemia progression (FIGS. 14C and D) and significantly prolonged survival in tumor mice (FIG. 14B).

Example 9: detection, isolation, amplification and engineering of human MAIT cells

Human MAIT cells are detected, isolated, expanded, and engineered using the methods described herein.

As shown in fig. 11, human MAIT cells were analyzed by flow cytometry.

As shown in fig. 12, the MAIT cells were flow sorted.

The MAIT cells were then activated and transduced with a CAR-expressing vector to produce CAR-MAIT cells, which were then expanded, as shown in FIG. 13.

Example 10: by stimulating PMBC expansionIncreasing MAIT cells

MAIT cells are a subset of congenital T cells, defined as CD3+TCRVa7.2+CD161+ cells, recognizing the MHC class I molecule MR1. Previous studies have shown that MAIT cells can be expanded in vitro, but require the presence of allogeneic feeder cells. However, one problem with this approach is that it is difficult to mass produce and quality control. In this study, the inventors have now developed a very novel and effective method for expanding MAIT cells in vitro by first stimulating PBMC with antigen (5-OP-RU) loaded MR1 tetramer beads or with 5-OP-RU alone, in vitro culture in the presence of a combination of cytokines (IL-2, IL-7, IL-15, IL-12, IL-18 and IL-23) for up to 6 days. MAIT cells were then isolated by MACS or FACS sorting, followed by further expansion by anti-CD 3/CD28 beads for CAR-based treatment as described in the previous examples.

Materials and methods

PBMC isolation

PBMCs were isolated from buffy coats of healthy blood donors by Ficoll-Hypaque density gradient centrifugation. An aliquot of isolated PBMCs was frozen and stored in liquid nitrogen for later use. Before starting the experiment, frozen PBMC stock solutions were thawed and cultured at 37 ℃ in RPMI medium supplemented with 10% FBS.

Preparation of MR1/5-OP-RU composite beads

MR1/5-OP-RU tetramer coated beads were prepared by using M-280dynabeads and streptavidin (from ThermoFisher) and biotinylated MR1 monomer. MR1 monomer loaded with 5-OP-RU was offered by Jim McCluskey doctor (university of Meerce Australia) friends. The beads were reacted with 5-OP-RU loaded MR1 monomer (5 ug/3x10 ⁷ Beads) were mixed by standing at 4℃for 12 hours on a shaking table. The excess unbound protein was removed by washing twice with PBS for 10 minutes. The prepared MR1 tetramer-coated beads were resuspended in PBS and stored at 4 ℃ for later use.

Enrichment of MAIT cells in PBMC

PBMC (per well)2x10 ⁵ Individual cells) were cultured in 96-well plates containing R10 medium (90% rpmi+10% fbs+1% penicillin/streptomycin+2 mM L-glutamine) in 37 ℃ incubator, stimulated by MR1/5-OP-RU complex coated beads (1:1 ratio of beads to cells) or purified 5-OP-RU antigen (10 nM) (supplied by Jeffrey Mak doctor, university of kunsland australia) and bound to different cytokines in vitro for 6 days. Cytokines IL-2 (100 IU/ml) (Roche), IL-7 (50 ng/ml) (Miltenyi), IL-15 (50 ng/ml) (Miltenyi), IL-12 (50 ng/ml) (Miltenyi), IL-18 (50 ng/ml) (ThermoFisher) and IL-23 (50 ng/ml) (Miltenyi) were added in 15 different combinations, numbered 1 to 15, as shown in the table in FIG. 15. On day 6, the expanded cells were collected and analyzed, and the percentage of MAIT cells was determined by flow cytometry, as described below.

FACS analysis of MAIT cell frequency in PBMC

Amplified PBMCs were surface marker stained with antibodies in the dark for 30 minutes. FITC-conjugated CD3 (clone BW264/56, miltenyi), PE-conjugated Va7.2 (clone 3C10, biolegend), APC-conjugated CD161 (clone DX12, BD) were used to label cells at a ratio of 1:100. MAIT cells are defined as CD3+Va7.2+CD161+ cells. Using LIVE/DEAD ^TM Dead cells were excluded from the Fixable Aqua dead cell staining kit (ThermoFisher). The stained cells were washed with 5 to 10 volumes of PBS, centrifuged at 500 Xg for 5 minutes, then resuspended in 200. Mu.l PBS and analyzed by flow cytometry. Flow cytometry results were analyzed by FlowJo.

Results

FIG. 15 shows the results of enrichment of MAIT cells in PBMC. PBMC were stimulated by either (i) bead to cell ratio 1:1 MR1/5-OP-RU composite beads, or (ii) 10nM of 5-OP-RU antigen, in the presence of different cytokines (IL-2, IL-7, IL-15, IL-12, IL-18 and IL-23) for 6 days as shown in the table. For example, condition 1 corresponds to IL-2 alone, condition 2 corresponds to IL-7 and IL-15, condition 3 corresponds to IL-2, IL-12 and IL-18, and so on.

The fold increase in MAIT cells was calculated by dividing the frequency of viable MAIT (CD3+Va7.2+CD161+) cells on day 6 by the original frequency of MAIT cells on day 0. The first five groups are marked with orange colors (i.e., conditions 1, 3, 11, 12, and 13).

It can be seen that the cytokine combinations of 1, 13, 12, 3 and 11 gave the highest fold increase of MAIT cells in PBMC for the MR1/5-OP-RU composite beads (donor 1). For the MR1/5-OP-RU composite beads (donor 2), the cytokine combinations of 12, 13, 1, 11 and 3 gave the highest fold increase of MAIT cells in PBMC.

It can be seen that the cytokine combinations of 3, 1, 12, 13 and 11 gave the highest fold increase of MAIT cells in PBMC for 5-OP-RU (donor 1). For 5-OP-RU (donor 2), the cytokine combinations of 8, 13, 12, 11 and 3 gave the highest fold increase of MAIT cells in PBMC.

From these data, it is evident that different cytokines and combinations of cytokines (IL-2, IL-7, IL-15, IL-12, IL-18 and IL-23) produce different degrees of stimulation, thereby increasing the enrichment of MAIT cells in PBMC. Overall, the combination of IL-12, IL-18 and IL-23 maximizes the fold increase in MAIT cells in PBMC.

Discussion of the invention

MAIT cells are a subset of congenital T cells, defined as CD3+TCRVa7.2+CD161+ cells, recognizing the MHC class I molecule MR1. Previous studies have shown that MAIT cells can be expanded in vitro, but require the presence of allogeneic feeder cells. However, one problem with this approach is that it is difficult to mass produce and quality control. As described above, the inventors have now developed a surprisingly effective method for in vitro expansion of MAIT cells, wherein an in vitro culture is performed in the presence of a combination of three cytokines (IL-12, IL-18 and IL-23) for up to 6 days, either by first stimulating PBMC with antigen (5-OP-RU) loaded MR1 tetrameric beads or with 5-OP-RU alone. MAIT cells are then isolated by MACS or FACS sorting, followed by further expansion by anti-CD 3/CD28 beads for CAR-based therapy.

Currently, there is no mature treatment for T cell lymphoma compared to B cell malignancy, the only potential treatment regimen is allogeneic Hematopoietic Stem Cell Transplantation (HSCT), but this approach itself has significant treatment-related mortality. Since most (> 95%) T cell lymphomas are derived from dominant T cell clones expressing a specific T Cell Receptor (TCR) gene (i.e. clone TCR-Vb chain) and the pan-T helper cell marker CD4, monoclonal antibodies directed against these markers have been used to treat T cell lymphomas, some of which lead to regression of part of the lymphoma in small clinical trials (d' Amore et al, 2010;Hagberg et al, 2005; kim et al, 2007).

Although CAR-T is a very effective treatment for B-cell malignancies by targeting the pan B-cell marker CD19, this approach has encountered significant obstacles when applied to the treatment of T-cell lymphomas. First, unlike B cell depletion, persistent T cell dysgenesis, particularly cd4+ T cell depletion, can lead to severe toxicity, such as opportunistic infections observed during chronic HIV infection. Second, the impaired T cell function associated with T cell lymphomas and the reduced normal T cell count resulting from dominant growth of T cell tumors cannot be used to produce autologous CAR-T cells, and thus allogeneic CAR-T cells are needed to treat T cell lymphomas. Finally, most T cell lymphomas are solid tumors associated with lymph nodes and skin tissue, which are difficult to treat with conventional CAR-T due to the low tissue infiltration capacity and poor tumor microenvironment.

To solve these problems, the inventors devised a CD4 antigen (CART 4) -targeted CAR containing tgfr and iC9 as safety switches to selectively eliminate CART4 transduced T cells after eradicating tumor cells, thereby restoring autologous hematopoietic stem cells or allogeneic HSCT to normal cd4+ T cells. Depletion of cd4+ T cells has been demonstrated to be safe and tolerable in the treatment of autoimmune diseases with anti-CD 4 antibodies (Hagberg et al, 2005; kim et al, 2007). CART4 transduced human T cells were able to kill cd4+ T cell lymphoma cell lines isolated from ATLL or CTCL patients in vitro and inhibit tumor growth in vivo in a mouse xenograft model. More importantly, these CART4+ T cells co-expressed CARs with tgfr (detected by anti-EGFR antibodies) and iC9 (determined by CID drug-induced apoptosis of CART4+ T cells in vitro and in vivo). Expression of tgfr can be used to monitor CART4+ T cell proliferation or to eliminate CART4+ T cells in vivo with anti-EGFR antibodies.

In general, normal T cells consist of a highly diverse pool of TCRs to maintain cellular immunity against pathogen infection. TCRs consist of heterodimers of a and b chains containing an N-terminal variable region and a C-terminal constant region. The TCR-Vb region (chain) is more polymorphic than TCR-Va and is commonly used to analyze immune responses or the clonality of T cell malignancies. Currently, the TCR-Vb chain family has 22 specific mAbs covering 75% of the TCR pool. Since most T cell lymphomas are derived from a single T cell clone expressing the same TCR Vb chain, CART of TCR-Vb chain defined for tumor clones while retaining the remaining normal T cell pool would be an ideal method to minimize opportunistic infections and without removal of CART cells after transfusion.

As proof of concept for anti-TCR-Vb immunotherapy, the inventors designed CARs that specifically target the TCR-Vb7.1 chain (cartvb7.1), and as a result demonstrated that cartvb7.1 transduced T cells could effectively eliminate TCR-vb7.1 positive tumor cells isolated from ATL patients, indicating that anti-TCR-Vb CARs could provide an alternative immunotherapy for T cell lymphomas.

Finally, the inventors investigated whether MAIT cells can be effector cells for CAR-based therapies, as MAIT cells have several advantages over traditional T cells, including:

(1) Since a highly conserved constant TCR is expressed during mammalian evolution, alloreactivity is low (i.e. induction of graft versus host disease, GVHD);

(2) Regulatory functions, including inhibition of GVHD in a mouse model;

(3) Activation induces the killing activity of GrB, perforin and GrA; and

(4) Tissue homing, for example, is distributed across intestinal mucosa, skin and lungs.

As described herein, CAR-transduced MAIT cells (CAR-MAIT) exhibit anti-tumor activity at least comparable to conventional CAR-T cells in vitro and in vivo. In summary, the inventors developed a novel CAR-MAIT based immunotherapy that could effectively treat T cell malignancies, by targeting the pan-T cell marker CD4 with switchable CAR-T, reducing the targeting/de-tumor toxicity of cytokine release syndrome or specific TCR-Vb chain (which is specific for malignant T cells), thus avoiding global immunosuppression. More importantly, CAR-MAIT cells may have the potential to develop the allogeneic CAR-based therapies needed to treat T cell lymphomas. If production is continued, i.e., large-scale in vitro amplification, they can be used for ready-to-use development. The inventors believe that CAR-MAIT may provide an effective new method of treatment not only for T cell malignancies, but also for tumors of other non-immune cell types.

Conclusion(s)

T cell therapies based on Chimeric Antigen Receptors (CARs) have met with great success in the treatment of B cell malignancies by targeting pan B cell specific antigens. However, similar strategies for T cell lymphomas have not been achieved to date, mainly due to the potentially serious toxicity associated with overall T cell depletion and low normal T cell dysfunction/frequency in T lymphomas compared to B cell malignancies. To overcome these limitations, the inventors devised a variety of novel CAR constructs specific for the pan-T cell marker (CD 4) or TCR-Vb isotype chain, comprising two safety switches: truncated epidermal growth factor receptor (tgfr) and inducible caspase 9 (iC 9). The inventors investigated whether mucosal-associated constant T (MAIT) cells with low alloreactivity would exhibit similar anti-tumor killing activity to conventional T cells after transduction with CAR constructs.

Surprisingly, CAR transduced T cells not only exhibit specific killing effects on cd4+ T lymphoma cells or TCR-Vb specific T leukemia clones isolated from patients, but also are eliminated after treatment with inducers in vitro and in vivo. Furthermore, the inventors have also demonstrated for the first time that CAR-MAIT cells are able to inhibit tumor growth as effectively as conventional T cells in vitro and in vivo. The study provides a new strategy for the treatment of T cell lymphomas.

Thus, the mucosa-associated constant T (MAIT) cells of the invention are a class of immune cells which are involved in a wide range of infectious and non-infectious diseases and which have unusual specificity for microbial riboflavin derivative antigens presented by the histocompatibility complex (MHC) class I protein MR1, which have been developed into a novel immunotherapy for the treatment of cancer patients enabling them to specifically recognize and attack T lymphomas by genetic engineering of the MAIT cells with Chimeric Antigen Receptors (CARs).

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Attachment paper

1. A mucosal associated constant T (MAIT) cell expressing a Chimeric Antigen Receptor (CAR).

2. The MAIT cell of clause 1, wherein the CAR-MAIT cell expresses a CAR that targets a CD4 antigen on a T cell.

3. The MAIT cell of clause 2, wherein the CAR pair comprises a sequence substantially as set forth in SEQ ID No:1 or a variant or fragment thereof.

4. The MAIT cell according to any one of the preceding claims, wherein said CAR-MAIT cell expresses a CAR targeting the T Cell Receptor (TCR) β chain variable region (Vbeta) on T cells, preferably any one of the Vbeta regions as shown in table 1.

5. The MAIT cell of clause 4, wherein the CAR targets multiple T Cell Receptor (TCR) β chain variable regions (Vbeta) on the T cell, preferably wherein the multiple Vbeta regions are selected from the group of Vbeta regions shown in table 1, optionally wherein the multiple TCR Vbeta regions are the same or different Vbeta regions.

6. The MAIT cell of clause 4 or 5, wherein the CAR targets one or more TCR Vbeta regions on a T cell selected from the group consisting of: vb1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb17 and Vb 20.

7. The MAIT cell of any one of clauses 4-6, wherein the CAR pair comprises a polypeptide substantially as set forth in SEQ id no:2 or a variant or fragment thereof.

8. The MAIT cell of any one of the preceding claims, wherein the CAR-MAIT cell comprises one or more coding sequences that allow the CAR-MAIT cell to be controllably or inductively depleted.

9. The MAIT cell of clause 8, wherein the one or more coding sequences encode an Epidermal Growth Factor Receptor (EGFR) or a truncated epidermal growth factor receptor (tgfr).

10. The MAIT cell of clause 8 or 9, wherein the one or more coding sequences encode inducible caspase 9 (iC 9).

11. The MAIT cells according to any of the preceding claims, wherein said MAIT cells are isolated from human Peripheral Blood Mononuclear Cells (PBMCs) by Magnetic Activated Cell Sorting (MACS) and/or Fluorescence Activated Cell Sorting (FACS), more preferably both MACS and FACS.

12. A nucleic acid construct comprising a promoter operably linked to a first coding sequence encoding an anti-CD 4 Chimeric Antigen Receptor (CAR) or an anti-T Cell Receptor (TCR) Vbeta CAR.

13. The construct of clause 12, wherein the promoter is a PGK promoter, optionally the promoter comprises a nucleotide sequence substantially as set forth in SEQ ID No:3 or a fragment or variant thereof.

14. The construct of clause 12 or 13, wherein the first coding sequence encodes an anti-CD 4 Chimeric Antigen Receptor (CAR), optionally wherein the CAR pair comprises a sequence substantially as set forth in SEQ ID No:1 or a variant or fragment thereof.

15. The construct of clause 14, wherein:

(i) The first coding sequence comprises a sequence encoding a polypeptide substantially as set forth in SEQ ID No:6 or a fragment or variant thereof;

(ii) The first coding sequence comprises a sequence substantially as set forth in SEQ ID No:7 or a fragment or variant thereof;

(iii) The first coding sequence comprises a sequence encoding a polypeptide substantially as set forth in SEQ ID No:8 or a fragment or variant thereof; and/or

(iv) The first coding sequence comprises a sequence substantially as set forth in SEQ ID No:9 or a fragment or variant thereof.

16. The construct of clause 12 or 13, wherein the first coding sequence encodes an anti-T Cell Receptor (TCR) Vbeta region CAR, optionally any Vbeta region listed in table 1.

17. The construct of clause 16, wherein the first coding sequence encodes a plurality of T Cell Receptor (TCR) β chain variable regions (Vbeta) CARs, preferably wherein the plurality of Vbeta regions is selected from the group of Vbeta regions shown in table 1.

18. The construct of clause 16 or 17, wherein the construct comprises a coding sequence encoding at least one CAR that targets one or more Vbeta regions of a TCR on a T cell selected from the group consisting of: vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20; optionally wherein the construct comprises a coding sequence encoding at least one CAR that targets at least two or three Vbeta regions of a TCR on a T cell selected from the group consisting of: vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20.

19. The construct of any one of clauses 16 to 18, wherein the CAR pair comprises a sequence substantially as set forth in SEQ ID No:2 or a variant or fragment thereof (preferably a TCR-Vbeta 7.1 chain).

20. The construct of any one of clauses 16 to 19, wherein:

(i) The first coding sequence comprises a sequence encoding a polypeptide substantially as set forth in SEQ ID No:12 or a fragment or variant thereof;

(ii) The first coding sequence comprises a sequence substantially as set forth in SEQ ID No:13 or a fragment or variant thereof;

(iii) The first coding sequence comprises a sequence encoding a polypeptide substantially as set forth in SEQ ID No:34 or a fragment or variant thereof; and/or

(iv) The first coding sequence comprises a sequence substantially as set forth in SEQ ID No:35 or a fragment or variant thereof.

21. The construct of any one of clauses 12 to 20, wherein the construct comprises a nucleotide sequence encoding a CD8a hinge and Transmembrane (TM) domain, optionally wherein the construct comprises a nucleotide sequence encoding a sequence substantially as set forth in SEQ ID No:14 or a fragment or variant thereof, and/or wherein the construct comprises a nucleotide sequence substantially as set forth in SEQ id no:15 or a fragment or variant thereof.

22. The construct according to any one of clauses 12 to 21, wherein the construct comprises a nucleotide sequence encoding an intracellular domain comprising the signaling domain of CD28, the signaling domain of 4-1BB and/or the CD3 zeta chain, wherein the signaling domain of CD28, the signaling domain of 4-1BB and the CD3 zeta chain are more preferred.

23. The construct of clause 22, wherein:

(i) The construct comprises a nucleic acid sequence encoding a polypeptide substantially as set forth in SEQ ID No:16 or a fragment or variant thereof;

(ii) The construct comprises a sequence substantially as set forth in SEQ ID No:17 or a fragment or variant thereof;

(iii) The construct comprises a nucleic acid sequence encoding a polypeptide substantially as set forth in SEQ ID No:18 or a fragment or variant thereof;

(iv) The construct comprises a sequence substantially as set forth in SEQ ID No:19 or a fragment or variant thereof;

(v) The construct comprises a nucleic acid sequence encoding a polypeptide substantially as set forth in SEQ ID No:20 or a fragment or variant thereof; and/or

(vi) The construct comprises a sequence substantially as set forth in SEQ ID No:21 or a fragment or variant thereof.

24. The construct according to any one of clauses 12 to 23, wherein the nucleic acid construct comprises a second coding sequence encoding at least one suicide protein, more preferably at least two suicide proteins.

25. The construct of clause 24, wherein the second coding sequence encodes: (i) An Epidermal Growth Factor Receptor (EGFR) or truncated epidermal growth factor receptor (tgfr); and/or (ii) inducible caspase 9 (iC 9).

26. The construct of clause 25, wherein:

(i) The construct comprises a nucleic acid sequence encoding a polypeptide substantially as set forth in SEQ ID No:22 or a fragment or variant thereof, optionally wherein the construct comprises a nucleotide sequence substantially as set forth in SEQ ID No:23 or a fragment or variant thereof; and/or

(ii) The construct comprises a nucleic acid sequence encoding a polypeptide substantially as set forth in SEQ ID No:24 or a fragment or variant thereof, optionally wherein the construct comprises a nucleotide sequence substantially as set forth in SEQ ID No:25 or a fragment or variant thereof.

27. The construct of any one of clauses 12 to 26, wherein the construct comprises a sequence encoding a polypeptide substantially as set forth in SEQ id no:29 or a fragment or variant thereof, optionally wherein the construct comprises a nucleotide sequence substantially as set forth in SEQ ID No:30 or a fragment or variant thereof.

28. The construct of any one of clauses 12 to 26, wherein the construct comprises a sequence encoding a polypeptide substantially as set forth in SEQ id no:31 or a fragment or variant thereof, optionally wherein the construct comprises a nucleotide sequence substantially as set forth in SEQ ID No:32 or a fragment or variant thereof.

29. An expression vector encoding the nucleic acid construct according to any one of clauses 12 to 28, optionally wherein the vector comprises a sequence substantially as set forth in SEQ ID No:33 or SEQ ID No:36 or a fragment or variant thereof.

30. A method of isolating a MAIT cell, the method comprising:

(i) Providing Peripheral Blood Mononuclear Cells (PBMCs); and

(ii) The PBMCs are subjected to Magnetic Activated Cell Sorting (MACS) and/or Fluorescent Activated Cell Sorting (FACS) to separate the MAIT cells therefrom.

31. A method of producing a CAR-MAIT cell, the method comprising:

(i) Providing Peripheral Blood Mononuclear Cells (PBMCs);

(ii) MACS and/or FACS are performed on PBMCs to isolate MAIT cells therefrom;

(iii) Activating the isolated MAIT cells, optionally by contacting them with an anti-CD 3 and/or anti-CD 28 antibody; and

(iv) Transduction of activated MAIT cells with nucleic acid encoding a CAR, thereby producing CAR-MAIT cells.

32. The method of clause 30 or 31, wherein the method comprises MACS and FACS of PBMCs to isolate the MAIT cells therefrom, optionally wherein the PBMCs are MACS followed by FACS.

33. The method of any one of clauses 30 to 32, wherein the isolated MAIT cells are activated with an anti-CD 3 antibody and an anti-CD 28 antibody.

34. The method of any one of clauses 31 to 33, wherein step (iv) comprises transducing a MAIT cell with a nucleic acid encoding a CAR by a virus or retrovirus, preferably wherein the nucleic acid encodes a CAR that targets (i) a CD4 antigen, or (ii) at least one or more TCR Vbeta regions on a T cell, preferably one or more TCR Vbeta regions shown in table 1, or one or more TCR Vbeta regions on a T cell selected from the group consisting of: vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20.

35. The method of any one of clauses 30 to 43, wherein the MAIT cells are transduced with the nucleic acid construct of any one of clauses 12 to 28 or the expression vector of clause 29.

36. The method of any one of clauses 30 to 35, wherein the method comprises expanding CAR-MAIT cells in a subsequent step after step (iv).

37. A CAR-MAIT cell obtained or obtainable by the method according to any one of items 30 to 36.

38. A pharmaceutical composition comprising the MAIT cells of any one of clauses 1-11 or clause 37 and a pharmaceutically acceptable excipient.

39. The MAIT cell according to any one of clauses 1 to 11 or 37 or the pharmaceutical composition according to clause 38 for use in therapy.

40. The MAIT cell according to any one of clauses 1 to 11 or 37 or the pharmaceutical composition according to clause 38 for use in (i) immunotherapy; (ii) treating, preventing or ameliorating cancer; (ii) treating, preventing or ameliorating a microbial infection; or (iv) treating, preventing or ameliorating an autoimmune disease.

41. The MAIT cell according to any one of clauses 1 to 11 or 37 or the pharmaceutical composition according to clause 38 for use according to clause 39 or 40, wherein for the treatment, prevention or amelioration of a T cell malignancy, optionally a solid tumor or a liquid tumor.

42. The MAIT cell according to any one of clauses 1 to 11 or 37 or the pharmaceutical composition according to clause 38 for the use according to clause 41, wherein the T cell malignancy is Peripheral T Cell Lymphoma (PTCL) or Cutaneous T Cell Lymphoma (CTCL).

43. The use of the MAIT cell of any one of clauses 1 to 11 or 37 or the pharmaceutical composition of clause 38 for the use of clause 41, wherein:

(i) The PTCL is a PTCL subtype selected from the group consisting of: adult T cell acute lymphocytic lymphoma or leukemia (ATL), enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous lipoteichthy lymphoma (SPTCL), precursor T cell acute lymphocytic lymphoma or leukemia, and angioimmunoblastic T cell lymphoma (AITL);

(ii) The CTCL is a CTCL subtype selected from the group consisting of: mycosis Fungoides (MF), sezary Syndrome (SS), and cd4+ small and medium polymorphic T cell lymphoproliferative diseases.

44. The use of the MAIT cells of any one of clauses 1 to 11 or 37 or the pharmaceutical composition of clause 38 for any one of clauses 40 to 43, wherein for treating, preventing or ameliorating a viral infection (e.g., HIV, HBV, HTLV, EBV, HPV), a bacterial infection (e.g., TB), or a fungal infection, or for treating, preventing or ameliorating an autoimmune disease, such as systemic lupus erythematosus, rheumatoid arthritis, or myasthenia gravis.

45. The use of the MAIT cell of any one of clauses 1 to 11 or 37 or the pharmaceutical composition of clause 38 for any one of clauses 40 to 44, wherein the use comprises triggering a sequence encoding a suicide protein, optionally wherein the method comprises administering an anti-EGFR antibody and/or a caspase-inducing drug (CID) to the subject.

46. A method of preparing the pharmaceutical composition of clause 38, comprising combining a therapeutically effective amount of the MAIT cells of any one of clauses 1-1 or clause 37 with a pharmaceutically acceptable excipient.

Sequence listing

<110> Imperial academy of technology Co., ltd

<120> chimeric antigen receptor T cells

<130> 100915PCT1

<150> 2105682.5

<151> 2021-04-21

<160> 36

<170> PatentIn version 3.5

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Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gln Leu

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Ala Leu Leu Pro Ala Ala Thr Gln Gly Lys Lys Val Val Leu Gly Lys

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Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser

35 40 45

Ile Gln Phe His Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn

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Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala

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Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile

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Lys Asn Leu Lys Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu

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Asp Gln Lys Glu Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn

115 120 125

Ser Asp Thr His Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu

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Ser Pro Pro Gly Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly

145 150 155 160

Lys Asn Ile Gln Gly Gly Lys Thr Leu Ser Val Ser Gln Leu Glu Leu

165 170 175

Gln Asp Ser Gly Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys

180 185 190

Val Glu Phe Lys Ile Asp Ile Val Val Leu Ala Phe Gln Lys Ala Ser

195 200 205

Ser Ile Val Tyr Lys Lys Glu Gly Glu Gln Val Glu Phe Ser Phe Pro

210 215 220

Leu Ala Phe Thr Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp

225 230 235 240

Gln Ala Glu Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu

245 250 255

Lys Asn Lys Glu Val Ser Val Lys Arg Val Thr Gln Asp Pro Lys Leu

260 265 270

Gln Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro Gln Ala Leu

275 280 285

Pro Gln Tyr Ala Gly Ser Gly Asn Leu Thr Leu Ala Leu Glu Ala Lys

290 295 300

Thr Gly Lys Leu His Gln Glu Val Asn Leu Val Val Met Arg Ala Thr

305 310 315 320

Gln Leu Gln Lys Asn Leu Thr Cys Glu Val Trp Gly Pro Thr Ser Pro

325 330 335

Lys Leu Met Leu Ser Leu Lys Leu Glu Asn Lys Glu Ala Lys Val Ser

340 345 350

Lys Arg Glu Lys Ala Val Trp Val Leu Asn Pro Glu Ala Gly Met Trp

355 360 365

Gln Cys Leu Leu Ser Asp Ser Gly Gln Val Leu Leu Glu Ser Asn Ile

370 375 380

Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile

385 390 395 400

Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile

405 410 415

Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met

420 425 430

Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro

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His Arg Phe Gln Lys Thr Cys Ser Pro Ile

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Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala

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Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met

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Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His

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Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu

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Met Phe Val Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro

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Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His

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Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Gln

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<211> 501

<212> DNA

<213> Artificial Sequence

<220>

<223> PGK promoter

<400> 3

gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg 60

gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc 120

gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca 180

ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac 240

gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt 300

ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg 360

gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 420

ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 480

cctcattctc cgggcctttc g 501

<210> 4

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Signalling peptide

<400> 4

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp

20

<210> 5

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> Signalling peptide

<400> 5

atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ttccacaggt 60

gac 63

<210> 6

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> First coding sequence (VL for CD4)

<400> 6

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 7

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> First coding sequence (VL for CD4)

<400> 7

gacattgtga tgactcagag ccccgacagc ctggccgtct cactgggcga aagggtgacc 60

atgaattgta aatcttctca gagcctgctg tacagtacaa accagaaaaa ttacctggcc 120

tggtatcagc agaaacccgg ccagagccct aagctgctga tctattgggc aagtacccga 180

gagtcaggag tgccagacag attctccggg tctggaagtg gcacagactt caccctgaca 240

attagctccg tgcaggccga ggacgtggct gtctactatt gccagcagta ctatagctac 300

cgaactttcg gcgggggaac caaactggaa atcaag 336

<210> 8

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> First coding sequence (VH for CD4)

<400> 8

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 366

<212> DNA

<213> Artificial Sequence

<220>

<223> First coding sequence (VH for CD4)

<400> 9

caggtgcagc tgcagcagtc cggaccagag gtggtcaaac ccggcgctag cgtcaaaatg 60

tcctgtaagg catctggcta cactttcacc tcttatgtga ttcactgggt cagacagaag 120

cctgggcagg gactggactg gatcgggtac attaacccat ataatgatgg aactgactac 180

gatgaaaagt ttaaaggcaa ggccacactg acttccgaca cctcaacaag cactgcttat 240

atggagctgt ctagtctgag gtctgaagac acagcagtgt actattgcgc ccgcgagaag 300

gataactacg ccactggcgc ttggtttgca tattggggcc aggggaccct ggtgacagtc 360

tcatcc 366

<210> 10

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> G4S linker sequence

<400> 10

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 11

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223> G4S linker sequence

<400> 11

gaggaggagg cagtggcgga ggagggtcag gaggaggagg aagc 44

<210> 12

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> First coding sequence (VL for TCR V-beta)

<400> 12

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Gly Ser Gly Phe Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Asn Tyr Trp Tyr Phe Asp Val Trp Gly Thr Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 13

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> First coding sequence (VL for TCR V-beta)

<400> 13

caagttcagc tgcaacagcc tggcgccgag cttgtgaaac ctggcgcctc tgtgaagatg 60

agctgcaagg cctccggcta caccttcacc agatactgga tcacctgggt caagcagagg 120

cctggacagg gactcgagtg gatcggcgat atctatcctg gctccggctt caccaagtac 180

aacgagaagt tcaagagcaa ggccacactg accgtggaca ccagcagcag cacagcctac 240

atgcagctgt ctagcctgac cagcgaggac agcgccgtgt actactgtgc tagagaaggc 300

ggcaactact ggtacttcga cgtgtggggc accggcacca cagtgacagt tagttct 357

<210> 14

<211> 83

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8a hinge and transmembrane domain

<400> 14

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly

50 55 60

Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn

65 70 75 80

His Arg Asn

<210> 15

<211> 249

<212> DNA

<213> Artificial Sequence

<220>

<223> CD8a hinge and transmembrane domain

<400> 15

ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc cagcgccgcg accaccaaca 60

ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 120

gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 180

cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaac 240

cacaggaac 249

<210> 16

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> CD28 signalling domain

<400> 16

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 17

<211> 123

<212> DNA

<213> Artificial Sequence

<220>

<223> CD28 signalling domain

<400> 17

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 18

<211> 47

<212> PRT

<213> Artificial Sequence

<220>

<223> 4-1BB signalling domain

<400> 18

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40 45

<210> 19

<211> 141

<212> DNA

<213> Artificial Sequence

<220>

<223> 4-1BB signalling domain

<400> 19

cgtttctctg ttgttaaacg gggcagaaag aagctcctgt atatattcaa acaaccattt 60

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 120

gaagaaggag gatgtgaact g 141

<210> 20

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3zeta chain

<400> 20

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 21

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> CD3zeta chain

<400> 21

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 22

<211> 357

<212> PRT

<213> Artificial Sequence

<220>

<223> Truncated EGFR

<400> 22

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 23

<211> 1071

<212> DNA

<213> Artificial Sequence

<220>

<223> Truncated EGFR

<400> 23

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccacgca aagtgtgtaa cggaataggt attggtgaat ttaaagactc actctccata 120

aatgctacga atattaaaca cttcaaaaac tgcacctcca tcagtggcga tctccacatc 180

ctgccggtgg catttagggg tgactccttc acacatactc ctcctctgga tccacaggaa 240

ctggatattc tgaaaaccgt aaaggaaatc acagggtttt tgctgattca ggcttggcct 300

gaaaacagga cggacctcca tgcctttgag aacctagaaa tcatacgcgg caggaccaag 360

caacatggtc agttttctct tgcagtcgtc agcctgaaca taacatcctt gggattacgc 420

tccctcaagg agataagtga tggagatgtg ataatttcag gaaacaaaaa tttgtgctat 480

gcaaatacaa taaactggaa aaaactgttt gggacctccg gtcagaaaac caaaattata 540

agcaacagag gtgaaaacag ctgcaaggcc acaggccagg tctgccatgc cttgtgctcc 600

cccgagggct gctggggccc ggaacccagg gactgcgtct cttgccggaa tgtcagccga 660

ggcagggaat gcgtggacaa gtgcaacctt ctggagggtg agccaaggga gtttgtggag 720

aactctgagt gcatacagtg ccacccagag tgcctgcctc aggccatgaa catcacctgc 780

acaggacggg gaccagacaa ctgtatccag tgtgcccact acattgacgg cccccactgc 840

gtcaagacct gcccggcagg agtcatggga gaaaacaaca ccctggtctg gaagtacgca 900

gacgccggcc atgtgtgcca cctgtgccat ccaaactgca cctacggatg cactgggcca 960

ggtcttgaag gctgtccaac gaatgggcct aagatcccgt ccatcgccac tgggatggtg 1020

ggggccctcc tcttgctgct ggtggtggcc ctggggatcg gcctcttcat g 1071

<210> 24

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> Inducible caspase-9 (iC9)

<400> 24

Met Leu Glu Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg

1 5 10 15

Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met

20 25 30

Leu Glu Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro

35 40 45

Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu

50 55 60

Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser

65 70 75 80

Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro

85 90 95

His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly

100 105 110

Gly Gly Ser Gly Val Asp Gly Phe Gly Asp Val Gly Ala Leu Glu Ser

115 120 125

Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys

130 135 140

Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly

145 150 155 160

Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg

165 170 175

Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr

180 185 190

Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His

195 200 205

Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln

210 215 220

Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys

225 230 235 240

Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys

245 250 255

Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly

260 265 270

Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu

275 280 285

Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln

290 295 300

Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro

305 310 315 320

Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val

325 330 335

Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp

340 345 350

Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu

355 360 365

Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met

370 375 380

Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser

385 390 395 400

Val Asp Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Asp

405 410

<210> 25

<211> 1242

<212> DNA

<213> Artificial Sequence

<220>

<223> Inducible caspase-9 (iC9)

<400> 25

atgctcgagg gagtgcaggt ggaaaccatc tccccaggag acgggcgcac cttccccaag 60

cgcggccaga cctgcgtggt gcactacacc gggatgcttg aagatggaaa gaaagttgat 120

tcctcccggg acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga 180

ggctgggaag aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct 240

ccagattatg cctatggtgc cactgggcac ccaggcatca tcccaccaca tgccactctc 300

gtcttcgatg tggagcttct aaaactggaa tctggcggtg gatccggagt cgacggattt 360

ggtgatgtcg gtgctcttga gagtttgagg ggaaatgcag atttggctta catcctgagc 420

atggagccct gtggccactg cctcattatc aacaatgtga acttctgccg tgagtccggg 480

ctccgcaccc gcactggctc caacatcgac tgtgagaagt tgcggcgtcg cttctcctcg 540

ctgcatttca tggtggaggt gaagggcgac ctgactgcca agaaaatggt gctggctttg 600

ctggagctgg cgcagcagga ccacggtgct ctggactgct gcgtggtggt cattctctct 660

cacggctgtc aggccagcca cctgcagttc ccaggggctg tctacggcac agatggatgc 720

cctgtgtcgg tcgagaagat tgtgaacatc ttcaatggga ccagctgccc cagcctggga 780

gggaagccca agctcttttt catccaggcc tgtggtgggg agcagaaaga ccatgggttt 840

gaggtggcct ccacttcccc tgaagacgag tcccctggca gtaaccccga gccagatgcc 900

accccgttcc aggaaggttt gaggaccttc gaccagctgg acgccatatc tagtttgccc 960

acacccagtg acatctttgt gtcctactct actttcccag gttttgtttc ctggagggac 1020

cccaagagtg gctcctggta cgttgagacc ctggacgaca tctttgagca gtgggctcac 1080

tctgaagacc tgcagtccct cctgcttagg gtcgctaatg ctgtttcggt gaaagggatt 1140

tataaacaga tgcctggttg ctttaatttc ctccggaaaa aacttttctt taaaacatca 1200

gtcgactatc cgtacgacgt accagactac gcactcgact aa 1242

<210> 26

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> P2A spacer sequence

<400> 26

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 27

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> P2A spacer sequence

<400> 27

ggatccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga agaaaacccc 60

ggtcct 66

<210> 28

<211> 592

<212> DNA

<213> Woodchuck hepatitis virus

<400> 28

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tg 592

<210> 29

<211> 1370

<212> PRT

<213> Artificial Sequence

<220>

<223> Nucleic acid construct (CART4)

<400> 29

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu

20 25 30

Ala Val Ser Leu Gly Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln

35 40 45

Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln

50 55 60

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr

65 70 75 80

Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val

100 105 110

Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val

145 150 155 160

Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

165 170 175

Phe Thr Ser Tyr Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly

180 185 190

Leu Asp Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr

195 200 205

Asp Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr

210 215 220

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

225 230 235 240

Val Tyr Tyr Cys Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp

245 250 255

Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala

260 265 270

Ala Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala

275 280 285

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

290 295 300

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

305 310 315 320

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

325 330 335

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

340 345 350

Asn His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr

355 360 365

Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln

370 375 380

Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser

385 390 395 400

Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

405 410 415

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

420 425 430

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

435 440 445

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

450 455 460

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

465 470 475 480

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

485 490 495

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

500 505 510

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

515 520 525

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

530 535 540

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala

545 550 555 560

Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro

565 570 575

Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro

580 585 590

His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly

595 600 605

Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys

610 615 620

His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro

625 630 635 640

Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro

645 650 655

Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu

660 665 670

Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu

675 680 685

Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser

690 695 700

Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu

705 710 715 720

Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu

725 730 735

Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly

740 745 750

Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala

755 760 765

Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly

770 775 780

Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg

785 790 795 800

Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe

805 810 815

Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln

820 825 830

Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln

835 840 845

Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala

850 855 860

Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala

865 870 875 880

Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr

885 890 895

Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser

900 905 910

Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala

915 920 925

Leu Gly Ile Gly Leu Phe Met Gly Ser Gly Ala Thr Asn Phe Ser Leu

930 935 940

Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Glu

945 950 955 960

Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro

965 970 975

Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp

980 985 990

Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe

995 1000 1005

Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val

1010 1015 1020

Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro

1025 1030 1035

Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro

1040 1045 1050

His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser

1055 1060 1065

Gly Gly Gly Ser Gly Val Asp Gly Phe Gly Asp Val Gly Ala Leu

1070 1075 1080

Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met

1085 1090 1095

Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys

1100 1105 1110

Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys

1115 1120 1125

Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu

1130 1135 1140

Val Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu

1145 1150 1155

Glu Leu Ala Gln Gln Asp His Gly Ala Leu Asp Cys Cys Val Val

1160 1165 1170

Val Ile Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro

1175 1180 1185

Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys

1190 1195 1200

Ile Val Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly

1205 1210 1215

Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys

1220 1225 1230

Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser

1235 1240 1245

Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly

1250 1255 1260

Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro Thr

1265 1270 1275

Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val

1280 1285 1290

Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu

1295 1300 1305

Asp Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser

1310 1315 1320

Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr

1325 1330 1335

Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe

1340 1345 1350

Phe Lys Thr Ser Val Asp Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1355 1360 1365

Leu Asp

1370

<210> 30

<211> 4113

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleic acid construct (CART4)

<400> 30

atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ttccacaggt 60

gacgacattg tgatgactca gagccccgac agcctggccg tctcactggg cgaaagggtg 120

accatgaatt gtaaatcttc tcagagcctg ctgtacagta caaaccagaa aaattacctg 180

gcctggtatc agcagaaacc cggccagagc cctaagctgc tgatctattg ggcaagtacc 240

cgagagtcag gagtgccaga cagattctcc gggtctggaa gtggcacaga cttcaccctg 300

acaattagct ccgtgcaggc cgaggacgtg gctgtctact attgccagca gtactatagc 360

taccgaactt tcggcggggg aaccaaactg gaaatcaagg gaggaggagg cagtggcgga 420

ggagggtcag gaggaggagg aagccaggtg cagctgcagc agtccggacc agaggtggtc 480

aaacccggcg ctagcgtcaa aatgtcctgt aaggcatctg gctacacttt cacctcttat 540

gtgattcact gggtcagaca gaagcctggg cagggactgg actggatcgg gtacattaac 600

ccatataatg atggaactga ctacgatgaa aagtttaaag gcaaggccac actgacttcc 660

gacacctcaa caagcactgc ttatatggag ctgtctagtc tgaggtctga agacacagca 720

gtgtactatt gcgcccgcga gaaggataac tacgccactg gcgcttggtt tgcatattgg 780

ggccagggga ccctggtgac agtctcatcc gcggccgcat tcgtgccggt cttcctgcca 840

gcgaagccca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 900

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 960

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 1020

gtccttctcc tgtcactggt tatcaccctt tactgcaacc acaggaacag gagtaagagg 1080

agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1140

aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc ccgtttctct 1200

gttgttaaac ggggcagaaa gaagctcctg tatatattca aacaaccatt tatgagacca 1260

gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1320

ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 1380

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1440

aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1500

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1560

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1620

accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcgg atccggagcc 1680

acgaacttct ctctgttaaa gcaagcagga gacgtggaag aaaaccccgg tcctatgctt 1740

ctcctggtga caagccttct gctctgtgag ttaccacacc cagcattcct cctgatccca 1800

cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 1860

acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 1920

gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 1980

attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 2040

aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 2100

ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 2160

aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 2220

acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 2280

agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 2340

ggctgctggg gcccggaacc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 2400

gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 2460

gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 2520

cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag 2580

acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 2640

ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt 2700

gaaggctgtc caacgaatgg gcctaagatc ccgtccatcg ccactgggat ggtgggggcc 2760

ctcctcttgc tgctggtggt ggccctgggg atcggcctct tcatgggatc tggagccacg 2820

aacttctctc tgttaaagca agcaggagac gtggaagaaa accccggtcc tatgctcgag 2880

ggagtgcagg tggaaaccat ctccccagga gacgggcgca ccttccccaa gcgcggccag 2940

acctgcgtgg tgcactacac cgggatgctt gaagatggaa agaaagttga ttcctcccgg 3000

gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa 3060

gaaggggttg cccagatgag tgtgggtcag agagccaaac tgactatatc tccagattat 3120

gcctatggtg ccactgggca cccaggcatc atcccaccac atgccactct cgtcttcgat 3180

gtggagcttc taaaactgga atctggcggt ggatccggag tcgacggatt tggtgatgtc 3240

ggtgctcttg agagtttgag gggaaatgca gatttggctt acatcctgag catggagccc 3300

tgtggccact gcctcattat caacaatgtg aacttctgcc gtgagtccgg gctccgcacc 3360

cgcactggct ccaacatcga ctgtgagaag ttgcggcgtc gcttctcctc gctgcatttc 3420

atggtggagg tgaagggcga cctgactgcc aagaaaatgg tgctggcttt gctggagctg 3480

gcgcagcagg accacggtgc tctggactgc tgcgtggtgg tcattctctc tcacggctgt 3540

caggccagcc acctgcagtt cccaggggct gtctacggca cagatggatg ccctgtgtcg 3600

gtcgagaaga ttgtgaacat cttcaatggg accagctgcc ccagcctggg agggaagccc 3660

aagctctttt tcatccaggc ctgtggtggg gagcagaaag accatgggtt tgaggtggcc 3720

tccacttccc ctgaagacga gtcccctggc agtaaccccg agccagatgc caccccgttc 3780

caggaaggtt tgaggacctt cgaccagctg gacgccatat ctagtttgcc cacacccagt 3840

gacatctttg tgtcctactc tactttccca ggttttgttt cctggaggga ccccaagagt 3900

ggctcctggt acgttgagac cctggacgac atctttgagc agtgggctca ctctgaagac 3960

ctgcagtccc tcctgcttag ggtcgctaat gctgtttcgg tgaaagggat ttataaacag 4020

atgcctggtt gctttaattt cctccggaaa aaacttttct ttaaaacatc agtcgactat 4080

ccgtacgacg taccagacta cgcactcgac taa 4113

<210> 31

<211> 1366

<212> PRT

<213> Artificial Sequence

<220>

<223> Nucleic acid construct (CARTVb7.1)

<400> 31

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Gly Lys Val Thr Leu Thr Cys Lys Ala Ser Gln

35 40 45

Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly

50 55 60

Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile

85 90 95

Ser Asn Leu Glu Pro Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr

100 105 110

Asp Asn Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

115 120 125

Thr Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly

145 150 155 160

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg

165 170 175

Tyr Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp

180 185 190

Ile Gly Asp Ile Tyr Pro Gly Ser Gly Phe Thr Lys Tyr Asn Glu Lys

195 200 205

Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala

210 215 220

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

225 230 235 240

Cys Ala Arg Glu Gly Gly Asn Tyr Trp Tyr Phe Asp Val Trp Gly Thr

245 250 255

Gly Thr Thr Val Thr Val Ser Ser Ala Ala Ala Ala Ala Phe Val Pro

260 265 270

Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro

275 280 285

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

290 295 300

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

305 310 315 320

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

325 330 335

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn

340 345 350

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

355 360 365

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

370 375 380

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser Val Val Lys Arg

385 390 395 400

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

405 410 415

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

420 425 430

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

435 440 445

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

450 455 460

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

465 470 475 480

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

485 490 495

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

500 505 510

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

515 520 525

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

530 535 540

His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser

545 550 555 560

Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu

565 570 575

Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe

580 585 590

Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe

595 600 605

Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn

610 615 620

Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg

625 630 635 640

Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp

645 650 655

Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala

660 665 670

Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile

675 680 685

Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val

690 695 700

Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser

705 710 715 720

Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn

725 730 735

Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys

740 745 750

Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val

755 760 765

Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg

770 775 780

Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp

785 790 795 800

Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser

805 810 815

Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile

820 825 830

Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr

835 840 845

Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly

850 855 860

Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys

865 870 875 880

His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu

885 890 895

Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly

900 905 910

Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly

915 920 925

Leu Phe Met Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala

930 935 940

Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Glu Gly Val Gln Val

945 950 955 960

Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln

965 970 975

Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val

980 985 990

Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys

995 1000 1005

Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser

1010 1015 1020

Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr

1025 1030 1035

Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu

1040 1045 1050

Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly Ser

1055 1060 1065

Gly Val Asp Gly Phe Gly Asp Val Gly Ala Leu Glu Ser Leu Arg

1070 1075 1080

Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly

1085 1090 1095

His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly

1100 1105 1110

Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg

1115 1120 1125

Arg Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp

1130 1135 1140

Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln

1145 1150 1155

Gln Asp His Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser

1160 1165 1170

His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr

1175 1180 1185

Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn Ile

1190 1195 1200

Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu

1205 1210 1215

Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe

1220 1225 1230

Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn

1235 1240 1245

Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe

1250 1255 1260

Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile

1265 1270 1275

Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val Ser Trp Arg Asp

1280 1285 1290

Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe

1295 1300 1305

Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg

1310 1315 1320

Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met Pro

1325 1330 1335

Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser

1340 1345 1350

Val Asp Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Asp

1355 1360 1365

<210> 32

<211> 4101

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleic acid construct (CARTVb7.1)

<400> 32

atggctctgc ctgttacagc tctgctgctg cctctggctc tgcttctgca tgccgccaga 60

cctgacatcc agatgacaca gagccctagc agcctgtctg cctctctcgg cggaaaagtg 120

accctgacat gcaaggccag ccaggacatc aacaagtata tcgcctggta tcagcacaag 180

cccggcaagg gacctagact gctgatccac tacaccagca cactgcagcc tggcatcccc 240

agcagatttt ctggcagcgg ctccggcaga gactacagct tcagcatcag caacctggaa 300

cctgaggacg tggccaccta ctactgcctg cagtacgaca acctgcggac ctttggcggc 360

ggaacaaagc tggaaatcaa gcggacagat ggcggaggcg gatcaggcgg cggaggaagc 420

ggtggcggag gatctcaagt tcagctgcaa cagcctggcg ccgagcttgt gaaacctggc 480

gcctctgtga agatgagctg caaggcctcc ggctacacct tcaccagata ctggatcacc 540

tgggtcaagc agaggcctgg acagggactc gagtggatcg gcgatatcta tcctggctcc 600

ggcttcacca agtacaacga gaagttcaag agcaaggcca cactgaccgt ggacaccagc 660

agcagcacag cctacatgca gctgtctagc ctgaccagcg aggacagcgc cgtgtactac 720

tgtgctagag aaggcggcaa ctactggtac ttcgacgtgt ggggcaccgg caccacagtg 780

acagttagtt ctgcggccgc ggccgcattc gtgccggtct tcctgccagc gaagcccacc 840

acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 900

ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 960

ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 1020

tcactggtta tcacccttta ctgcaaccac aggaacagga gtaagaggag caggctcctg 1080

cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1140

ccctatgccc caccacgcga cttcgcagcc tatcgctccc gtttctctgt tgttaaacgg 1200

ggcagaaaga agctcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1260

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1320

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1380

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1440

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1500

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1560

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1620

tacgacgccc ttcacatgca ggccctgccc cctcgcggat ccggagccac gaacttctct 1680

ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ctatgcttct cctggtgaca 1740

agccttctgc tctgtgagtt accacaccca gcattcctcc tgatcccacg caaagtgtgt 1800

aacggaatag gtattggtga atttaaagac tcactctcca taaatgctac gaatattaaa 1860

cacttcaaaa actgcacctc catcagtggc gatctccaca tcctgccggt ggcatttagg 1920

ggtgactcct tcacacatac tcctcctctg gatccacagg aactggatat tctgaaaacc 1980

gtaaaggaaa tcacagggtt tttgctgatt caggcttggc ctgaaaacag gacggacctc 2040

catgcctttg agaacctaga aatcatacgc ggcaggacca agcaacatgg tcagttttct 2100

cttgcagtcg tcagcctgaa cataacatcc ttgggattac gctccctcaa ggagataagt 2160

gatggagatg tgataatttc aggaaacaaa aatttgtgct atgcaaatac aataaactgg 2220

aaaaaactgt ttgggacctc cggtcagaaa accaaaatta taagcaacag aggtgaaaac 2280

agctgcaagg ccacaggcca ggtctgccat gccttgtgct cccccgaggg ctgctggggc 2340

ccggaaccca gggactgcgt ctcttgccgg aatgtcagcc gaggcaggga atgcgtggac 2400

aagtgcaacc ttctggaggg tgagccaagg gagtttgtgg agaactctga gtgcatacag 2460

tgccacccag agtgcctgcc tcaggccatg aacatcacct gcacaggacg gggaccagac 2520

aactgtatcc agtgtgccca ctacattgac ggcccccact gcgtcaagac ctgcccggca 2580

ggagtcatgg gagaaaacaa caccctggtc tggaagtacg cagacgccgg ccatgtgtgc 2640

cacctgtgcc atccaaactg cacctacgga tgcactgggc caggtcttga aggctgtcca 2700

acgaatgggc ctaagatccc gtccatcgcc actgggatgg tgggggccct cctcttgctg 2760

ctggtggtgg ccctggggat cggcctcttc atgggatctg gagccacgaa cttctctctg 2820

ttaaagcaag caggagacgt ggaagaaaac cccggtccta tgctcgaggg agtgcaggtg 2880

gaaaccatct ccccaggaga cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg 2940

cactacaccg ggatgcttga agatggaaag aaagttgatt cctcccggga cagaaacaag 3000

ccctttaagt ttatgctagg caagcaggag gtgatccgag gctgggaaga aggggttgcc 3060

cagatgagtg tgggtcagag agccaaactg actatatctc cagattatgc ctatggtgcc 3120

actgggcacc caggcatcat cccaccacat gccactctcg tcttcgatgt ggagcttcta 3180

aaactggaat ctggcggtgg atccggagtc gacggatttg gtgatgtcgg tgctcttgag 3240

agtttgaggg gaaatgcaga tttggcttac atcctgagca tggagccctg tggccactgc 3300

ctcattatca acaatgtgaa cttctgccgt gagtccgggc tccgcacccg cactggctcc 3360

aacatcgact gtgagaagtt gcggcgtcgc ttctcctcgc tgcatttcat ggtggaggtg 3420

aagggcgacc tgactgccaa gaaaatggtg ctggctttgc tggagctggc gcagcaggac 3480

cacggtgctc tggactgctg cgtggtggtc attctctctc acggctgtca ggccagccac 3540

ctgcagttcc caggggctgt ctacggcaca gatggatgcc ctgtgtcggt cgagaagatt 3600

gtgaacatct tcaatgggac cagctgcccc agcctgggag ggaagcccaa gctctttttc 3660

atccaggcct gtggtgggga gcagaaagac catgggtttg aggtggcctc cacttcccct 3720

gaagacgagt cccctggcag taaccccgag ccagatgcca ccccgttcca ggaaggtttg 3780

aggaccttcg accagctgga cgccatatct agtttgccca cacccagtga catctttgtg 3840

tcctactcta ctttcccagg ttttgtttcc tggagggacc ccaagagtgg ctcctggtac 3900

gttgagaccc tggacgacat ctttgagcag tgggctcact ctgaagacct gcagtccctc 3960

ctgcttaggg tcgctaatgc tgtttcggtg aaagggattt ataaacagat gcctggttgc 4020

tttaatttcc tccggaaaaa acttttcttt aaaacatcag tcgactatcc gtacgacgta 4080

ccagactacg cactcgacta a 4101

<210> 33

<211> 10348

<212> DNA

<213> Artificial Sequence

<220>

<223> Vector (CART4: CAR4-tEGFR-iC9)

<400> 33

tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60

ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120

gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180

acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240

ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc 300

gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc 360

ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata 420

aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca 480

gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga 540

cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg 600

tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt 660

actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa 720

cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga 780

cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag 840

acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa 900

gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg 960

tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct 1020

taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga 1080

gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag 1140

acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc 1200

cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc 1260

cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg 1320

ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag 1380

ccctcactcc ttctctaggc gccggaatta gatctctcga ggttaacgaa ttctaccggg 1440

taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca 1500

cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc 1560

aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga 1620

agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc 1680

tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg 1740

gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg 1800

ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg 1860

gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct 1920

cattctccgg gcctttcgac ctgcagccca agccaccatg gagacagaca cactcctgct 1980

atgggtgctg ctgctctggg ttccaggttc cacaggtgac gacattgtga tgactcagag 2040

ccccgacagc ctggccgtct cactgggcga aagggtgacc atgaattgta aatcttctca 2100

gagcctgctg tacagtacaa accagaaaaa ttacctggcc tggtatcagc agaaacccgg 2160

ccagagccct aagctgctga tctattgggc aagtacccga gagtcaggag tgccagacag 2220

attctccggg tctggaagtg gcacagactt caccctgaca attagctccg tgcaggccga 2280

ggacgtggct gtctactatt gccagcagta ctatagctac cgaactttcg gcgggggaac 2340

caaactggaa atcaagggag gaggaggcag tggcggagga gggtcaggag gaggaggaag 2400

ccaggtgcag ctgcagcagt ccggaccaga ggtggtcaaa cccggcgcta gcgtcaaaat 2460

gtcctgtaag gcatctggct acactttcac ctcttatgtg attcactggg tcagacagaa 2520

gcctgggcag ggactggact ggatcgggta cattaaccca tataatgatg gaactgacta 2580

cgatgaaaag tttaaaggca aggccacact gacttccgac acctcaacaa gcactgctta 2640

tatggagctg tctagtctga ggtctgaaga cacagcagtg tactattgcg cccgcgagaa 2700

ggataactac gccactggcg cttggtttgc atattggggc caggggaccc tggtgacagt 2760

ctcatccgcg gccgcattcg tgccggtctt cctgccagcg aagcccacca cgacgccagc 2820

gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga 2880

ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga 2940

tatctacatc tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat 3000

caccctttac tgcaaccaca ggaacaggag taagaggagc aggctcctgc acagtgacta 3060

catgaacatg actccccgcc gccccgggcc cacccgcaag cattaccagc cctatgcccc 3120

accacgcgac ttcgcagcct atcgctcccg tttctctgtt gttaaacggg gcagaaagaa 3180

gctcctgtat atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga 3240

tggctgtagc tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt 3300

cagcaggagc gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct 3360

caatctagga cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga 3420

gatgggggga aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa 3480

agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa 3540

ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct 3600

tcacatgcag gccctgcccc ctcgcggatc cggagccacg aacttctctc tgttaaagca 3660

agcaggagac gtggaagaaa accccggtcc tatgcttctc ctggtgacaa gccttctgct 3720

ctgtgagtta ccacacccag cattcctcct gatcccacgc aaagtgtgta acggaatagg 3780

tattggtgaa tttaaagact cactctccat aaatgctacg aatattaaac acttcaaaaa 3840

ctgcacctcc atcagtggcg atctccacat cctgccggtg gcatttaggg gtgactcctt 3900

cacacatact cctcctctgg atccacagga actggatatt ctgaaaaccg taaaggaaat 3960

cacagggttt ttgctgattc aggcttggcc tgaaaacagg acggacctcc atgcctttga 4020

gaacctagaa atcatacgcg gcaggaccaa gcaacatggt cagttttctc ttgcagtcgt 4080

cagcctgaac ataacatcct tgggattacg ctccctcaag gagataagtg atggagatgt 4140

gataatttca ggaaacaaaa atttgtgcta tgcaaataca ataaactgga aaaaactgtt 4200

tgggacctcc ggtcagaaaa ccaaaattat aagcaacaga ggtgaaaaca gctgcaaggc 4260

cacaggccag gtctgccatg ccttgtgctc ccccgagggc tgctggggcc cggaacccag 4320

ggactgcgtc tcttgccgga atgtcagccg aggcagggaa tgcgtggaca agtgcaacct 4380

tctggagggt gagccaaggg agtttgtgga gaactctgag tgcatacagt gccacccaga 4440

gtgcctgcct caggccatga acatcacctg cacaggacgg ggaccagaca actgtatcca 4500

gtgtgcccac tacattgacg gcccccactg cgtcaagacc tgcccggcag gagtcatggg 4560

agaaaacaac accctggtct ggaagtacgc agacgccggc catgtgtgcc acctgtgcca 4620

tccaaactgc acctacggat gcactgggcc aggtcttgaa ggctgtccaa cgaatgggcc 4680

taagatcccg tccatcgcca ctgggatggt gggggccctc ctcttgctgc tggtggtggc 4740

cctggggatc ggcctcttca tgggatctgg agccacgaac ttctctctgt taaagcaagc 4800

aggagacgtg gaagaaaacc ccggtcctat gctcgaggga gtgcaggtgg aaaccatctc 4860

cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg 4920

gatgcttgaa gatggaaaga aagttgattc ctcccgggac agaaacaagc cctttaagtt 4980

tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt 5040

gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc 5100

aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaatc 5160

tggcggtgga tccggagtcg acggatttgg tgatgtcggt gctcttgaga gtttgagggg 5220

aaatgcagat ttggcttaca tcctgagcat ggagccctgt ggccactgcc tcattatcaa 5280

caatgtgaac ttctgccgtg agtccgggct ccgcacccgc actggctcca acatcgactg 5340

tgagaagttg cggcgtcgct tctcctcgct gcatttcatg gtggaggtga agggcgacct 5400

gactgccaag aaaatggtgc tggctttgct ggagctggcg cagcaggacc acggtgctct 5460

ggactgctgc gtggtggtca ttctctctca cggctgtcag gccagccacc tgcagttccc 5520

aggggctgtc tacggcacag atggatgccc tgtgtcggtc gagaagattg tgaacatctt 5580

caatgggacc agctgcccca gcctgggagg gaagcccaag ctctttttca tccaggcctg 5640

tggtggggag cagaaagacc atgggtttga ggtggcctcc acttcccctg aagacgagtc 5700

ccctggcagt aaccccgagc cagatgccac cccgttccag gaaggtttga ggaccttcga 5760

ccagctggac gccatatcta gtttgcccac acccagtgac atctttgtgt cctactctac 5820

tttcccaggt tttgtttcct ggagggaccc caagagtggc tcctggtacg ttgagaccct 5880

ggacgacatc tttgagcagt gggctcactc tgaagacctg cagtccctcc tgcttagggt 5940

cgctaatgct gtttcggtga aagggattta taaacagatg cctggttgct ttaatttcct 6000

ccggaaaaaa cttttcttta aaacatcagt cgactatccg tacgacgtac cagactacgc 6060

actcgactaa acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 6120

aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 6180

attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 6240

tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 6300

gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 6360

ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 6420

ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt 6480

ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 6540

ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 6600

cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 6660

cctatcgata aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc 6720

cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaataca 6780

taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca gaatatgggc 6840

caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc 6900

cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc 6960

ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct 7020

tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggc 7080

gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt gtatccaata aaccctcttg 7140

cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac 7200

tacccgtcag cgggggtctt tcatgggtaa cagtttcttg aagttggaga acaacattct 7260

gagggtagga gtcgaatatt aagtaatcct gactcaatta gccactgttt tgaatccaca 7320

tactccaata ctcctgaaat agttcattat ggacagcgca gaagagctgg ggagaattaa 7380

ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 7440

caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 7500

cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 7560

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 7620

ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 7680

ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 7740

agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 7800

taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 7860

cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 7920

tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 7980

gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 8040

gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 8100

tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 8160

gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 8220

cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 8280

aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 8340

tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 8400

ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 8460

attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 8520

ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 8580

tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 8640

aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 8700

acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 8760

aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 8820

agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 8880

ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 8940

agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 9000

tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 9060

tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 9120

attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 9180

taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 9240

aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 9300

caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 9360

gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 9420

cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 9480

tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 9540

acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 9600

gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 9660

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 9720

cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat 9780

tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 9840

ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 9900

ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 9960

ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggcgcaag gaatggtgca 10020

tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg 10080

aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg 10140

atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg 10200

tagaggcgat tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg 10260

actcaacaat atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt 10320

ttatttagtc tccagaaaaa ggggggaa 10348

<210> 34

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> First coding sequence (VH for TCR V-beta)

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Leu Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Arg Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Asp

100 105

<210> 35

<211> 327

<212> DNA

<213> Artificial Sequence

<220>

<223> First coding sequence (VH for TCR V-beta)

<400> 35

gacatccaga tgacacagag ccctagcagc ctgtctgcct ctctcggcgg aaaagtgacc 60

ctgacatgca aggccagcca ggacatcaac aagtatatcg cctggtatca gcacaagccc 120

ggcaagggac ctagactgct gatccactac accagcacac tgcagcctgg catccccagc 180

agattttctg gcagcggctc cggcagagac tacagcttca gcatcagcaa cctggaacct 240

gaggacgtgg ccacctacta ctgcctgcag tacgacaacc tgcggacctt tggcggcgga 300

acaaagctgg aaatcaagcg gacagat 327

<210> 36

<211> 10336

<212> DNA

<213> Artificial Sequence

<220>

<223> Vector (CARTVb7.1: CARTVb7.1-tEGFR-iC9)

<400> 36

tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60

ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120

gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180

acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240

ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc 300

gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc 360

ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata 420

aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca 480

gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga 540

cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg 600

tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt 660

actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa 720

cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga 780

cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag 840

acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa 900

gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg 960

tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct 1020

taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga 1080

gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag 1140

acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc 1200

cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc 1260

cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg 1320

ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag 1380

ccctcactcc ttctctaggc gccggaatta gatctctcga ggttaacgaa ttctaccggg 1440

taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca 1500

cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc 1560

aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga 1620

agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc 1680

tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg 1740

gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg 1800

ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg 1860

gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct 1920

cattctccgg gcctttcgac ctgcagccca agccaccatg gctctgcctg ttacagctct 1980

gctgctgcct ctggctctgc ttctgcatgc cgccagacct gacatccaga tgacacagag 2040

ccctagcagc ctgtctgcct ctctcggcgg aaaagtgacc ctgacatgca aggccagcca 2100

ggacatcaac aagtatatcg cctggtatca gcacaagccc ggcaagggac ctagactgct 2160

gatccactac accagcacac tgcagcctgg catccccagc agattttctg gcagcggctc 2220

cggcagagac tacagcttca gcatcagcaa cctggaacct gaggacgtgg ccacctacta 2280

ctgcctgcag tacgacaacc tgcggacctt tggcggcgga acaaagctgg aaatcaagcg 2340

gacagatggc ggaggcggat caggcggcgg aggaagcggt ggcggaggat ctcaagttca 2400

gctgcaacag cctggcgccg agcttgtgaa acctggcgcc tctgtgaaga tgagctgcaa 2460

ggcctccggc tacaccttca ccagatactg gatcacctgg gtcaagcaga ggcctggaca 2520

gggactcgag tggatcggcg atatctatcc tggctccggc ttcaccaagt acaacgagaa 2580

gttcaagagc aaggccacac tgaccgtgga caccagcagc agcacagcct acatgcagct 2640

gtctagcctg accagcgagg acagcgccgt gtactactgt gctagagaag gcggcaacta 2700

ctggtacttc gacgtgtggg gcaccggcac cacagtgaca gttagttctg cggccgcggc 2760

cgcattcgtg ccggtcttcc tgccagcgaa gcccaccacg acgccagcgc cgcgaccacc 2820

aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc 2880

agcggcgggg ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg 2940

ggcgcccttg gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg 3000

caaccacagg aacaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac 3060

tccccgccgc cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt 3120

cgcagcctat cgctcccgtt tctctgttgt taaacggggc agaaagaagc tcctgtatat 3180

attcaaacaa ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg 3240

ccgatttcca gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc 3300

agacgccccc gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg 3360

aagagaggag tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa 3420

gccgagaagg aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc 3480

ggaggcctac agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg 3540

cctttaccag ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc 3600

cctgccccct cgcggatccg gagccacgaa cttctctctg ttaaagcaag caggagacgt 3660

ggaagaaaac cccggtccta tgcttctcct ggtgacaagc cttctgctct gtgagttacc 3720

acacccagca ttcctcctga tcccacgcaa agtgtgtaac ggaataggta ttggtgaatt 3780

taaagactca ctctccataa atgctacgaa tattaaacac ttcaaaaact gcacctccat 3840

cagtggcgat ctccacatcc tgccggtggc atttaggggt gactccttca cacatactcc 3900

tcctctggat ccacaggaac tggatattct gaaaaccgta aaggaaatca cagggttttt 3960

gctgattcag gcttggcctg aaaacaggac ggacctccat gcctttgaga acctagaaat 4020

catacgcggc aggaccaagc aacatggtca gttttctctt gcagtcgtca gcctgaacat 4080

aacatccttg ggattacgct ccctcaagga gataagtgat ggagatgtga taatttcagg 4140

aaacaaaaat ttgtgctatg caaatacaat aaactggaaa aaactgtttg ggacctccgg 4200

tcagaaaacc aaaattataa gcaacagagg tgaaaacagc tgcaaggcca caggccaggt 4260

ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg gaacccaggg actgcgtctc 4320

ttgccggaat gtcagccgag gcagggaatg cgtggacaag tgcaaccttc tggagggtga 4380

gccaagggag tttgtggaga actctgagtg catacagtgc cacccagagt gcctgcctca 4440

ggccatgaac atcacctgca caggacgggg accagacaac tgtatccagt gtgcccacta 4500

cattgacggc ccccactgcg tcaagacctg cccggcagga gtcatgggag aaaacaacac 4560

cctggtctgg aagtacgcag acgccggcca tgtgtgccac ctgtgccatc caaactgcac 4620

ctacggatgc actgggccag gtcttgaagg ctgtccaacg aatgggccta agatcccgtc 4680

catcgccact gggatggtgg gggccctcct cttgctgctg gtggtggccc tggggatcgg 4740

cctcttcatg ggatctggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga 4800

agaaaacccc ggtcctatgc tcgagggagt gcaggtggaa accatctccc caggagacgg 4860

gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga 4920

tggaaagaaa gttgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa 4980

gcaggaggtg atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc 5040

caaactgact atatctccag attatgccta tggtgccact gggcacccag gcatcatccc 5100

accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaatctg gcggtggatc 5160

cggagtcgac ggatttggtg atgtcggtgc tcttgagagt ttgaggggaa atgcagattt 5220

ggcttacatc ctgagcatgg agccctgtgg ccactgcctc attatcaaca atgtgaactt 5280

ctgccgtgag tccgggctcc gcacccgcac tggctccaac atcgactgtg agaagttgcg 5340

gcgtcgcttc tcctcgctgc atttcatggt ggaggtgaag ggcgacctga ctgccaagaa 5400

aatggtgctg gctttgctgg agctggcgca gcaggaccac ggtgctctgg actgctgcgt 5460

ggtggtcatt ctctctcacg gctgtcaggc cagccacctg cagttcccag gggctgtcta 5520

cggcacagat ggatgccctg tgtcggtcga gaagattgtg aacatcttca atgggaccag 5580

ctgccccagc ctgggaggga agcccaagct ctttttcatc caggcctgtg gtggggagca 5640

gaaagaccat gggtttgagg tggcctccac ttcccctgaa gacgagtccc ctggcagtaa 5700

ccccgagcca gatgccaccc cgttccagga aggtttgagg accttcgacc agctggacgc 5760

catatctagt ttgcccacac ccagtgacat ctttgtgtcc tactctactt tcccaggttt 5820

tgtttcctgg agggacccca agagtggctc ctggtacgtt gagaccctgg acgacatctt 5880

tgagcagtgg gctcactctg aagacctgca gtccctcctg cttagggtcg ctaatgctgt 5940

ttcggtgaaa gggatttata aacagatgcc tggttgcttt aatttcctcc ggaaaaaact 6000

tttctttaaa acatcagtcg actatccgta cgacgtacca gactacgcac tcgactaaac 6060

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 6120

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 6180

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 6240

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 6300

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 6360

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 6420

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 6480

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 6540

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 6600

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tatcgataaa 6660

ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt 6720

tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaatacata actgagaata 6780

gagaagttca gatcaaggtt aggaacagag agacagcaga atatgggcca aacaggatat 6840

ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt 6900

cccgccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga 6960

aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg 7020

cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc 7080

cgatagactg cgtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg 7140

acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg 7200

ggggtctttc atgggtaaca gtttcttgaa gttggagaac aacattctga gggtaggagt 7260

cgaatattaa gtaatcctga ctcaattagc cactgttttg aatccacata ctccaatact 7320

cctgaaatag ttcattatgg acagcgcaga agagctgggg agaattaatt cgtaatcatg 7380

gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 7440

cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 7500

gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 7560

cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 7620

tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 7680

aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 7740

gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 7800

ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 7860

ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 7920

gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 7980

ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 8040

cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 8100

cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 8160

gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 8220

aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 8280

tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 8340

gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 8400

tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 8460

gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 8520

tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 8580

ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 8640

ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 8700

tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 8760

aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 8820

gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 8880

gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 8940

ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 9000

gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 9060

gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 9120

gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 9180

tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 9240

gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 9300

agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 9360

aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 9420

ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 9480

gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta 9540

agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 9600

tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 9660

cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 9720

tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt 9780

gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 9840

ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 9900

attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 9960

gttttcccag tcacgacgtt gtaaaacgac ggcgcaagga atggtgcatg caaggagatg 10020

gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc 10080

atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca 10140

gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggcgatta 10200

gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac tcaacaatat 10260

caccagctga agcctataga gtacgagcca tagataaaat aaaagatttt atttagtctc 10320

cagaaaaagg ggggaa 10336

Claims (39)

1. A method of isolating a MAIT cell, the method comprising:

(i) Providing Peripheral Blood Mononuclear Cells (PBMCs); and

(ii) The PBMCs are subjected to Magnetic Activated Cell Sorting (MACS) and/or Fluorescent Activated Cell Sorting (FACS) to separate the MAIT cells therefrom.

2. A method of producing a CAR-MAIT cell, the method comprising:

(i) Providing Peripheral Blood Mononuclear Cells (PBMCs);

(ii) MACS and/or FACS are performed on PBMCs to isolate MAIT cells therefrom;

(iii) Activating the isolated MAIT cells, optionally by contacting them with an anti-CD 3 and/or anti-CD 28 antibody; and

(iv) Transduction of activated MAIT cells with nucleic acid encoding a CAR, thereby producing CAR-MAIT cells.

3. The method of any one of the preceding claims, wherein the method comprises stimulating PBMCs prior to MACS and/or FACS of PBMCs.

4. The method of claim 3, wherein the stimulating step comprises contacting PBMCs: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and/or (b) a cytokine; preferably, the stimulating step comprises contacting PBMCs: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and (b) a cytokine.

5. The method of claim 4, wherein the cytokine is an interleukin.

6. The method of claim 4 or 5, wherein the cytokine is one or more interleukins selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-23, or any combination thereof.

7. The method of claim 6, wherein the one or more interleukins comprise: (i) IL-2; (ii) IL-12 and IL-18; (iii) IL-2, IL-12 and IL-18; (iv) IL-12, IL-18 and IL-23; (v) IL-2, IL-12, IL-18 and IL-23, or (vi) IL-7, IL-15, IL-12 and IL-18.

8. The method of claim 6 or 7, wherein the one or more interleukins comprises a combination of IL-12, IL-18 and IL-23.

9. The method of any one of the preceding claims, wherein the method comprises MACS and FACS of PBMCs to isolate MAIT cells therefrom.

10. The method according to any one of the preceding claims, wherein the PBMCs are MACS followed by FACS.

11. The method of any one of the preceding claims, wherein the isolated MAIT cells are activated with an anti-CD 3 antibody.

12. The method of any one of the preceding claims, wherein the isolated MAIT cells are activated with an anti-CD 28 antibody.

13. The method of any one of claims 2 to 12, wherein step (iv) comprises transducing a MAIT cell with a nucleic acid encoding a Chimeric Antigen Receptor (CAR), optionally said transduction is performed by a virus or a retrovirus.

14. The method of claim 13, wherein the nucleic acid encodes a CAR that targets a CD4 antigen on a T cell.

15. The method of claim 13, wherein the nucleic acid encodes a CAR that targets at least one or more TCR Vbeta regions on a T cell.

16. The method of claim 15, wherein the one or more TCR Vbeta regions are (i) TCR Vbeta regions shown in table 1, and/or (ii) are selected from the group consisting of: vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb8, vb 12, vb 13.1, vb 17 and Vb 20.

17. The method of any one of the preceding claims, wherein the method comprises expanding CAR-MAIT cells in a subsequent step following step (iv).

18. The method of claim 17, wherein the step of amplifying the CAR-MAIT cells comprises harvesting the transduced CAR-MAIT cells one or two days after transduction; optionally wherein the harvested cells are contacted with an interleukin, preferably IL-2; optionally wherein the interleukin is in R10 medium.

19. A CAR-MAIT cell obtained or obtainable by the method according to any one of the preceding claims.

20. A mucosal associated constant T (MAIT) cell expressing a Chimeric Antigen Receptor (CAR).

21. The MAIT cell of claim 19 or 20, wherein the CAR-MAIT cell expresses a CAR that targets a CD4 antigen on a T cell, optionally wherein the CAR pair comprises a polypeptide substantially as set forth in SEQ ID No:1 or a variant or fragment thereof.

22. The MAIT cell of claim 19 or 20, wherein the CAR-MAIT cell expresses a CAR targeting the T Cell Receptor (TCR) β chain variable region (Vbeta) on a T cell; optionally (i) any one of the Vbeta regions as shown in table 1, or (ii) a plurality of T Cell Receptor (TCR) β chain variable regions (Vbeta) on T cells, optionally wherein the plurality of Vbeta regions is selected from a set of Vbeta regions shown in table 1, optionally wherein the plurality of TCR Vbeta regions are the same or different Vbeta regions.

23. The MAIT cell of claim 22, wherein the CAR targets one or more TCR Vbeta regions on a T cell selected from the group consisting of: vb 1, vb 2, vb 3, vb 5.1, vb 7.1, vb 8, vb 12, vb 13.1, vb 17 and Vb 20, optionally wherein the CAR pair comprises a sequence substantially as set forth in SEQ ID No:2 or a variant or fragment thereof.

24. The MAIT cell of any one of the preceding claims, wherein the CAR-MAIT cell comprises one or more coding sequences that allow the CAR-MAIT cell to be controllably or inductively eliminated.

25. The MAIT cell of claim 24, wherein the one or more coding sequences encode an Epidermal Growth Factor Receptor (EGFR) or a truncated epidermal growth factor receptor (tgfr), optionally wherein the one or more coding sequences comprise a sequence encoding a polypeptide substantially as set forth in SEQ ID No:22, or a fragment or variant thereof, and/or comprises a nucleotide sequence substantially as set forth in SEQ ID No:23 or a fragment or variant thereof.

26. The MAIT cell of claim 24 or 25, wherein the one or more coding sequences encode an inducible caspase 9 (iC 9), optionally wherein the one or more coding sequences comprise a sequence encoding a polypeptide substantially as set forth in SEQ ID No:24, or a fragment or variant thereof, and/or comprises a nucleotide sequence substantially as set forth in SEQ ID No:25 or a fragment or variant thereof.

27. A pharmaceutical composition comprising the MAIT cells of any one of claims 19 to 26 and a pharmaceutically acceptable excipient.

28. The MAIT cell according to any one of claims 19 to 26 or the pharmaceutical composition according to claim 27 for use in therapy.

29. The MAIT cell according to any one of claims 19 to 26 or the pharmaceutical composition according to claim 27 for use in (i) immunotherapy; (ii) treating, preventing or ameliorating cancer; (ii) treating, preventing or ameliorating a microbial infection; or (iv) treating, preventing or ameliorating an autoimmune disease.

30. The use of a MAIT cell according to any one of claims 19 to 26 or a pharmaceutical composition according to claim 27 for the treatment, prevention or amelioration of a T cell malignancy, optionally a solid or liquid tumor.

31. The MAIT cell according to any one of claims 19 to 26 or the pharmaceutical composition according to claim 27 for use according to claim 30, wherein the T cell malignancy is Peripheral T Cell Lymphoma (PTCL) or Cutaneous T Cell Lymphoma (CTCL).

32. The use of a MAIT cell according to any one of claims 19 to 26 or a pharmaceutical composition according to claim 27 for the use according to claim 31, wherein:

(i) The PTCL is a PTCL subtype selected from the group consisting of: adult T cell acute lymphocytic lymphoma or leukemia (ATL), enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous lipoteichthy lymphoma (SPTCL), precursor T cell acute lymphocytic lymphoma or leukemia, and angioimmunoblastic T cell lymphoma (AITL); and/or

(ii) The CTCL is a CTCL subtype selected from the group consisting of: mycosis Fungoides (MF), sezary Syndrome (SS), and cd4+ small and medium polymorphic T cell lymphoproliferative diseases.

33. The MAIT cell according to any one of claims 19 to 26 or the pharmaceutical composition according to claim 27 for use according to claim 29, wherein for the treatment, prevention or amelioration of (i) viral infections, optionally HIV, HBV, HTLV, EBV or HPV, (ii) bacterial infections, optionally TB, or (iii) fungal infections; or for the treatment, prevention or amelioration of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis or myasthenia gravis.

34. The use of the MAIT cell of any one of claims 19 to 26 or the pharmaceutical composition of claim 27 for any one of claims 28 to 33, wherein the use comprises triggering a sequence encoding a suicide protein, optionally wherein the method comprises administering an anti-EGFR antibody and/or a caspase-inducing drug (CID) to a subject.

35. A method of preparing the pharmaceutical composition of claim 27, the method comprising combining a therapeutically effective amount of the MAIT cells of any one of claims 19 to 26 with a pharmaceutically acceptable excipient.

36. A method of stimulating MAIT cells in a PBMC culture, the method comprising contacting the PBMC culture with: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and/or (b) one or more interleukins selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-23, or any combination thereof.

37. The method of claim 36, wherein the method comprises contacting a PBMC culture with: (a) an antigen comprising MR1/5-OP-RU or 5-OP-RU; and (b) one or more interleukins selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-23, or any combination thereof.

38. The method of claim 36 or 37, wherein the one or more interleukins is IL-12, IL-18 and/or IL-23.

39. The method of claim 38, wherein the one or more interleukins are IL-12, IL-18, and IL-23.